MetAP-2 inhibitor polymersomes for therapeutic administration

ABSTRACT

Described herein are MetAP-2 inhibitors and compositions and formulations thereof, and more particularly compositions and formulations of MetAP-2 inhibitors wherein the MetAP-2 inhibitor is associated with a block copolymer comprising a hydrophilic polymer moiety and a hydrophobic polymer moiety. The present invention also relates to compositions and formulations comprising MetAP-2 inhibitors for oral administration or administration via routes such as topical or ocular administration. The present invention also provides methods to treat conditions associated with or related to the over-expression or over-activity of MetAP-2 by administering the compositions and formulations comprising MetAP-2 inhibitors as disclosed herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of co-pending U.S.application Ser. No. 12/648,078 filed on Dec. 28, 2009, which ofcontinuation-in-part of the International Application Serial No.PCT/US2008/68367, filed on Jun. 26, 2008, which claims benefit under 35U.S.C. §119(e) of the U.S. Provisional Application No. 60/937,198 filedJun. 26, 2007, and U.S. Provisional Application No. 61/054,595 filed May20, 2008, the contents of which are incorporated herein by reference intheir entireties.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.:W81XWH-05-1-0115 awarded by the Department of Defense. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to compositions and methods forthe treatment of angiogenic diseases and disorders.

BACKGROUND

Emerging data strongly implicates a ubiquitous eukaryotic enzyme“methionine aminopeptidase type 2” (MetAP-2) in many diseases (Kass2007, Yeh 2005, Bo 2004, Bringardner 2008, Zhang 2001, Watanabe 2006).While MetAP-2's role is not yet fully understood, it is clear thatexpression and activity of MetAP-2, including, but not necessarilylimited to over-expression or over-activity of MetAP-2, is correlatedwith disease progression and stage of disease (Salvukumar 2006).

It is a widely accepted hypothesis that tumor growth isangiogenesis-dependent, which is supported by both biological andpharmacological evidence and confirmed by genetic evidence whichprovides a scientific basis for current clinical trials of angiogenesisinhibitors. Increased tumor angiogenesis and elevated levels ofproangiogenic factors such as vascular endothelial growth factor(VEGF/VPF), basic fibroblast growth factor (bFGF), and interleukin-8(IL-8) correlate with decreased survival and increased risk of relapsein studies of patients with malignant solid tumors. The importance ofangiogenesis is further supported by the observation that antiangiogenicagents inhibit tumor growth in a variety of animal models.

In the U.S. there are currently more than 30 angiogenesis inhibitors invarious clinical trials for late-stage cancer. One of these angiogenesisinhibitors, O-(chloracetyl-carbamoyl) fumagillol (TNP-470), is a lowmolecular weight synthetic compound. Fumagillol is the alcohol derivedfrom the hydrolysis of fumagillin³, a compound secreted by the fungusAspergillus fumigatus fresenius. TNP-470 is a potent endothelialinhibitor in vitro³⁰. Recently, TNP-470 has been tested as a potentialnew anticancer agent. TNP-470 is also referred to as AGM-1470, an analogof fumagillin, is among the most potent inhibitors of angiogenesis. Theanti-angiogenic properties of the TNP-470 were tested on different tumormodels in animals, and in clinical trials with a variety of malignanciessuch as prostate, breast, lung and cervical cancer.

In animal models, TNP-470 has the broadest anticancer spectrum of anyknown agent^(32,31). TNP-470 inhibited the growth of murine tumors up to91%, human tumors up to 100% and metastatic tumors up to 100% in mice(reviewed in ref 31). In most studies, mice were treated at the sameoptimal dose of 30 mg/kg subcutaneously every other day. In clinicaltrials TNP-470 has shown evidence of antitumor activity when used as asingle agent, with a number of objective responses reported withrelapsed and refractory malignancies^(17,19,23). It has also shownpromise when used in combination with conventional chemotherapy^(20,34),although many patients experience neurotoxicity (malaise, rare seizures,asthenia, anxiety and dysphoria)^(20,21,22, 23) at high chemotherapeuticdoses where antitumor activity has been seen. In addition to itsanti-angiogenic activities, TNP-470 also inhibits or prevents vascularleakage.

Methionine aminopeptidase-2 (MetAP-2) has been identified as the targetof anti-angiogenic fumagillol derivatives (e.g., TNP-470).⁴¹ The crystalstructure of fumagillin complexed with MetAP-2 was reported by Liu etal.⁴²

TNP-470 is generally administered via injections, for exampleintramuscular or intravenous delivery systems or by a continuousintravenous infusion given every few days. TNP-470 is also typicallyadministered at high doses for a specific period of time at infrequenttime intervals, for example TNP-470 is administered at high doses as achemotherapeutic. TNP-470 is not generally administered orally.

SUMMARY

The present invention relates, in part, to compositions and formulationscomprising a MetAP-2 inhibitor and to compositions and formulationscomprising a MetAP-2 inhibitor associated with a block copolymer,wherein the block copolymer comprises a hydrophobic and hydrophilicmoiety. In some embodiments, the MetAP-2 inhibitor is associated with ablock copolymer comprising a hydrophobic moiety, wherein the hydrophobicmoiety is part of a block copolymer comprising hydrophilic andhydrophobic moieties. In some embodiments the block copolymer comprisinghydrophilic and hydrophobic moieties forms a micelle.

The MetAP-2 inhibitor-comprising compositions and formulations can beused in the treatment of a disease or disorder involving or associatedwith MetAP-2 expression, including, but not necessarily limited toMetAP-2 overexpression or over-activity. Accordingly, the presentinvention relates to a method to treat a condition wherein the conditioninvolves or requires MetAP-2 activity for its pathology, comprisingadministering a composition comprising a MetAP-2 inhibitor formulated asdescribed herein.

One aspect of the present invention relates to a composition comprisinga formulation of an irreversible MetAP-2 inhibitor, for example afumagillol analog or derivative that has anti-proliferative activity,where the formulation comprises the MetAP-2 inhibitor associated with ablock copolymer comprising a hydrophilic polymer moiety and ahydrophobic polymer moiety. In one embodiment, the irreversibleinhibitor is, for example, a member of the bengamide class of MetAP-2inhibitors, where the formulation is associated with a block copolymercomprising a hydrophilic polymer moiety and a hydrophobic polymermoiety.

Another aspect of the present invention relates to a compositioncomprising a formulation of reversible inhibitors, for example bestatinclass inhibitors or 3-amino-2-hydroxyamides and related hydroxyamidesand acylhydrazines, where the formulation is associated with a blockcopolymer comprising a hydrophilic polymer moiety and a hydrophobicpolymer moiety.

In some embodiments, the block copolymer is a diblock copolymer, forexample, the fumagillin analogs/derivatives or bengamide analogs thereofare associated with the hydrophobic moiety of a block copolymer such asa diblock copolymer. In such instances, the fumagillin analog/derivativeor bengamide analog is associated with the hydrophobic moiety of suchdiblock copolymer.

In some embodiments, the formulation comprises a micelle comprising theblock copolymer associated with the MetAP-2 reversible or irreversibleinhibitors.

The invention thus encompasses at least the following aspects andembodiments.

In one aspect, described herein is a composition comprising a methionineaminopeptidase-2 (MetAP-2) inhibitor formulation havinganti-proliferative activity, wherein the formulation comprises a MetAP-2inhibitor covalently linked to a block copolymer comprising ahydrophilic polymer moiety and a hydrophobic polymer moiety.

In one embodiment of this and all other aspects described herein, theMetAP-2 inhibitor is a fumagillol derivative.

In another embodiment of this and all other aspects described herein,the MetAP-2 inhibitor is an irreversible MetAP-2 inhibitor.

In another embodiment of this and all other aspects described herein,MetAP-2 inhibitor is a reversible MetAP-2 inhibitor.

In another embodiment of this and all other aspects described herein,block copolymer is a diblock copolymer.

In another embodiment of this and all other aspects described herein,MetAP-2 inhibitor is covalently linked with the hydrophobic moiety ofthe block copolymer.

In another embodiment of this and all other aspects described herein,the hydrophobic polymer moiety of the block copolymer is selected fromthe group consisting of poly(d,L-lactic acid), poly(caprolactone) (PCL),and poly(propylene oxide).

In another embodiment of this and all other aspects described herein,the hydrophobic moiety is a poly(d,L-lactic acid) (PLA) polymer.

In another embodiment of this and all other aspects described herein,the hydrophobic polymer moiety is 1-15 kDa. For example, the hydrophobicpolymer moiety can be between 1-10 kDa, 1-8 kDa, 1-5 kDa, 1-3 kDa, 3-15kDa, 5-15 kDa, 8-15 kDa, 10-15 kDa, 12-15 kDa, 2-12 kDa, or 4-10 kDa,6-8 kDa in size. In another embodiment of this and all other aspectsdescribed herein, the hydrophobic polymer is approximately 2 kDa.

In another embodiment of this and all other aspects described herein,the hydrophilic moiety is a poly(ethylene glycol) (PEG) polymer.

In another embodiment of this and all other aspects described herein,the hydrophilic polymer moiety is between 1-15 kDa. For example, ahydrophilic polymer moiety useful in the compositions as describedherein can be between 1-10 kDa, 1-8 kDa, 1-5 kDa, 1-3 kDa, 3-15 kDa,5-15 kDa, 8-15 kDa, 10-15 kDa, 12-15 kDa, 2-12 kDa, 4-10 kDa, 6-8 kDa insize. In another embodiment of this and all other aspects describedherein, the hydrophilic polymer is approximately 2 kDa.

In another embodiment of this and all other aspects described herein,the polymer is a diblock copolymer comprising a PEG-PLA diblockcopolymer having hydrophilic PEG and hydrophobic PLA moieties.

In another embodiment of this and all other aspects described herein,the formulation is formulated for oral administration.

In another embodiment of this and all other aspects described herein,the formulation is formulated for topical administration.

In another embodiment of this and all other aspects described herein,the formulation is formulated for administration by oral, IV,peritoneal, injected (e.g., subcutaneous, intramuscular, etc.), eyedropor ocular, suppository, topical, pulmonary, including inhaled, and nasalroutes, among others, and includes a reversible or irreversible MetAP-2inhibitor.

In another embodiment of this and all other aspects described herein,the anti-proliferative activity is an anti-tumor activity or an activitywhich normalizes vascular permeability.

In another embodiment of this and all other aspects described herein,the MetAP-2 inhibitor has anti-angiogenic activity and is useful in thepreparation of a medicament for the treatment of anangiogenesis-mediated condition and/or in a method of treating anangiogenesis-mediated condition.

In another embodiment of this and all aspects described herein relatingto fumagillol derivatives having anti-proliferative and/oranti-angiogenic activity, the fumagillol derivative comprises aderivative selected from the group consisting of6-O—(N-chloroacetylcarbamoyl)fumagillol (TNP-470),6-O-(4-methoxyaniline)acetyl fumagillol;6-O-(3,4,5-trimethexyaniline)acetyl fumagillol;6-O-(4-(N,N-dimethylethoxy)aniline)acetyl fumagillol;6-O-(cyclopropylamino)acetyl fumagillol; 6-O-(cyclobutylamino)acetylfumagillol;4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1cyclohexanol; 4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol.

In another embodiment of this and all aspects described herein relatingto fumagillol derivatives having anti-proliferative and/oranti-angiogenic activity, the fumagillol derivative comprises6-O—(N-chloroacetylcarbamoyl)fumagillol (i.e., TNP-470).

In another embodiment of this and all aspects described herein, theMetAP-2 inhibitor is selected from the group consisting of a bengamide,a sulphonamide MetAP-2 inhibitor compound, a bestatin, a3-amino-2-hydroxyamide MetAP-2 inhibitor compound, a hydroxyamideMetAP-2 inhibitor compound, an acylhydrazine MetAP-2 inhibitor compound,ovacillin, a reversible MetAP-2 inhibitor and an irreversible MetAP-2inhibitor.

In another aspect, described herein is a method of treating a conditionin which MetAP-2 activity is required for or involved in the pathologyof the condition, the method comprising administering a MetAP-2inhibitor formulation comprising a MetAP-2 inhibitor havinganti-proliferative activity, covalently linked to a block copolymercomprising a hydrophilic polymer moiety and a hydrophobic polymermoiety.

In one embodiment of this and all aspects described herein, thecondition is an angiogenesis-mediated condition. Angiogenesis mediatedconditions include, as non-limiting examples, cancer, metastatic tumors,ascites formation, psoriasis, age-related macular degeneration (AMD),thyroid hyperplasia, preeclampsia, rheumatoid arthritis andosteo-arthritis, Alzheimer's disease, obesity, pleural effusion,atherosclerosis, endometriosis, diabetic/other retinopathies, ocularneovascularizations such as neovascular glaucoma and cornealneovascularization, and IL-2 therapy associated edema and abnormalvascular proliferation of other types.

In one embodiment of this and all aspects described herein, thecondition involving MetAP-2 activity comprises a tumor activity.

In one embodiment of this and all aspects described herein, thecondition is selected from the group consisting of cancer, metastatictumors, psoriasis, age-related macular degeneration (AMD), thyroidhyperplasia, preeclampsia, rheumatoid arthritis and osteo-arthritis,Alzheimer's disease, obesity, pleural effusion, atherosclerosis,endometriosis, diabetic/other retinopathies, ocular neovascularizations,IL-2 therapy associated edema and other edemas, malaria, SARS, HIV,herpes, lupus, IPF, COPD, asthma, cystic fibrosis, transplant rejection,allergic reaction, multiple sclerosis, bacterial infection, viralinfection, and conditions involving or characterized by vascularhyperpermeability, inflammation, and spinal injury.

In another aspect, described herein is a method of making a diblockcopolymer composition comprising a MetAP-2 inhibitor that hasanti-proliferative activity, the method comprising conjugating theMetAP-2 inhibitor to a diblock copolymer comprising a hydrophilicpolymer moiety and a hydrophobic polymer moiety.

In one embodiment of this and all aspects described herein, the MetAP-2inhibitor is a fumagillol derivative that has anti-proliferative and/oranti-angiogenic activity.

In another aspect, described herein is a diblock copolymer compositioncomprising a MetAP-2 inhibitor, the composition produced by the methodsdescribed herein. In one embodiment, the MetAP-2 inhibitor is afumagillol derivative that has anti-proliferative activity and/oranti-angiogenic activity. In one embodiment, the fumagillol derivativecomprises 6-O—(N-chloroacetylcarbamoyl)fumagillol (TNP-470) and itsmetabolites.

In another aspect, described herein is the use of any of the MetAP-2inhibitor block copolymer formulations described herein in themanufacture of a medicament for the treatment of a condition that reliesupon or involves MetAP-2 activity for its pathology.

In one embodiment of this and all aspects described herein, thecondition comprises a tumor activity.

In another embodiment of this and all aspects described herein, thecondition is selected from the group consisting of cancer, metastatictumors, psoriasis, age-related macular degeneration (AMD), thyroidhyperplasia, preeclampsia, rheumatoid arthritis and osteo-arthritis,Alzheimer's disease, obesity, pleural effusion, atherosclerosis,endometriosis, diabetic/other retinopathies, ocular neovascularizations,IL-2 therapy associated edema and other edemas, malaria, SARS, HIV,herpes, lupus, IPF, COPD, asthma, cystic fibrosis, transplant rejection,allergic reaction, multiple sclerosis, bacterial infection, viralinfection, fungal infection and conditions involving or characterized byabnormal vasculature or vascular hyperpermeability, inflammation andspinal injury.

In one embodiment of this and all aspects described herein, is a methodof treating ocular neovascularization in a subject in need thereof, themethod comprising administering a MetAP-2 inhibitor formulationcomprising a MetAP-2 inhibitor having anti-angiogenic activity,associated with a block copolymer comprising a hydrophilic polymermoiety and a hydrophobic polymer moiety.

In another embodiment of this and all aspects described herein, is amethod of treating ocular neovascularization in a subject in needthereof, the method comprising administering a composition comprising aformulation of a fumagillol derivative that retains anti-angiogenicactivity, the formulation comprising the fumagillol derivative isassociated with a block copolymer comprising a hydrophilic polymermoiety and a hydrophobic polymer moiety. Examples of ocularneovascularization include but are not limited to AMD, ARMD,proliferative diabetic retinopathy (PDR), and retinopathy of prematurity(ROP). Encompasseh herein are eye diseases which involvesneovascularization, infection and edema.

In another aspect, described herein is a method of making a diblockcopolymer micelle comprising a MetAP-2 inhibitor, the method comprisingconjugating the MetAP-2 inhibitor to a diblock copolymer comprising ahydrophilic polymer moiety and a hydrophobic polymer moiety and formingmicelles of the resulting conjugate.

In another aspect, described herein is the production of a diblockcopolymer micelle comprising a MetAP-2 inhibitor.

Other aspects of the invention are disclosed infra.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows NMR spectra for a block copolymer and a block copolymerconjugate with TNP-470. Differences in NMR pattern verify the binding ofthe ethylendiamine and the TNP-470 to the polymer.

FIG. 2 shows amine levels of mPEG-PLA-en polymer before and afterTNP-470 binding. TNBSA reaction was performed as described in theExamples; levels of 450 nm absorption indicate the levels of freeamines.

FIG. 3 shows TNP-470 polymersomes' size ranges. A typical DLS graphshowing diameter distribution of TNP-470 polymersomes.

FIG. 4A shows inhibition of human umbilical vein epithelial cell (HUVEC)proliferation by MetAP-2 inhibitor polymersomes. Differentconcentrations of TNP-470 micelles (comprising between 62.5-1000 nM ofTNP-470 equivalent) showed a cytostatic effect on HUVECs as measured byWST-1 (*p<0.0005).

FIG. 4B shows that HUVEC proliferation was not affected by polymericmicelles without MetAP-2 inhibitor. No significant differences inproliferation were detected, even with a double concentration of carrierthan that of the MetAP-2 inhibitor polymersomes.

FIG. 5A shows the effect of MetAP-2 inhibitor polymersomes on tumorvolume. Tumor volume, measured during 14-18 days of daily oraladministration of TNP-470 polymeric micelles to C57/BL mice (*p<0.05).

FIG. 5B shows the effect of TNP-470 polymersomes on mice weights. Micewere weighed q.o.d, the results are an average weight of mice in eachgroup.

FIG. 6A shows a diagram of a conjugation reaction between TNP-470 andmodified mPEG-PLA.

FIG. 6B TEM images of Lodamin dispersed in water, the sphericalstructure of the micelles are shown at different time points postincubation in water bar=10 nm.

FIG. 6C TNP-470 release from Lodamin during a 30 d period as determinedby HPLC; Micelles were incubated in gastric fluid pH=1.2 (▪) or PBSpH=7.4 ( ) and analysis of the released TNP-470 was done in duplicate.The results are presented as means±SD.

FIG. 7A shows flow cytometry analysis of uptake of Rhodamine labeledmPEG-PLA micelles by HUVEC (FL2high) at 0 min, 20 min and 24 h ofincubation with micelle. Incubation after 2, 4 and 7 h showed a similarpattern as after 24 h of incubation (not shown).

FIG. 7B shows inhibition of HUVEC proliferation by differentconcentrations of Lodamin 50-1,000 nM TNP-470 equivalent. Emptypolymeric micelles were also added as a control (n=8, *P<0.0005).

FIG. 7C shows the HUVEC growth curve (●) of cells treated q.o.d. withLodamin (60 nM TNP-470 equivalent) (□) Untreated cells and (⋄) Vehicletreated cells.

FIG. 7D shows the quantification of neovascularization area in thecornea in a Corneal Micropocket assay (n=10, mean±SD). Inhibition ofangiogenesis in Lodamin treated mouse (15 mg/kg q.d.) with respect tocontrol.

FIG. 8A shows the fluorescent signal of tissue extracts and in serum ofmice treated with Lodamin. Mice (n=3) were given a single dose of oral6-coumarin labeled polymeric micelles (150 μl 30 mg/ml). Data show thevalues of the three different mice, autofluorescence was omitted bysubtracting the fluorescent signal of tissue extracts from an untreatedmouse. The percent of labeled cells was measured for each organ.

FIG. 8B shows the levels of fluorescent signals in mouse serum asmeasured after different time points post oral administration. Theresults are presented as the concentration of micelles calculated bystandard calibration curve.

FIG. 8C shows the percentage of FL2^(high) positive cells as isolatedfrom the designated organs (ratio of numbers FL2^(high) cells of treatedtumors to those of control mouse).

FIG. 8D shows the FACS analysis graphs of single-cell suspensions fromthree representative organs (tumor, liver, brain) taken from a mousebearing Lewis lung carcinoma after controlled enzymatic degradation. TheFL2^(high) represents those cells that contain the mPEG-PLA-rhodaminemicelles.

FIG. 9A shows the effect of free or conjugated TNP-470 on establishedprimary tumor-Lewis lung carcinoma tumors: effect of 30 mg/kg. q.o.d. offree TNP-470 (▪) given orally, compared to equivalent dose of Lodamin(x) or water ( ), (n=5 mice per group, *P<0.05).

FIG. 9B shows the Lewis lung carcinoma volume during 18 d of differentfrequencies and doses of Lodamin: 30 mg/kg. q.o.d. (x), 15 mg/kg q.o.d.(∘), 15 mg/kg q.d. (●), and water (□) by gavage (n=5 mice per group,*P<0.05.

FIG. 9C shows the effect of the vehicle, empty mPEG-PLA micelles, onLewis lung carcinoma (n=5 mice per group, *P<0.05).

FIG. 9D shows the effect of Lodamin given at a dose of 15 mg/kg q d. (●)on B16/F10 murine melanoma tumor in C57Bl/6J; water was given as control(□) (n=5 mice per group, *P<0.05).

FIG. 9E shows some representative Lewis lung carcinoma and B16/F10tumors removed from mice at day 18 post treatment with oral Lodamin at30 mg/kg q.o.d., and 15 mg/kg e.d. respectively, and from controluntreated mice.

FIG. 9F shows the neurotoxcity of Lodamin on treated mice (10 d, 30mg/kg q.o.d.) compared to mice treated with subcutaneous (30 mg/kgq.o.d.) free TNP-470 or water given by gavage. Balance beam test wasquantified by foot-slip errors and the numbers of slips per meter arepresented (n=4-5 mice per group). *P<0.05, **P<0.01, ***P<0.0001(results are mean±SE).

FIG. 9G shows the mice's weight on day 1 and day 18 post treatment withoral polymeric micelle TNP-470 (Lodamin).

FIG. 10A shows the livers removed from Lodamin-treated or untreated micewith B16/F10 tumors, 20 d post cell injection. Control livers wereenlarged with wide-spread macroscopic malignant nodules and extensivecirrhosis.

FIG. 10B shows the spleens removed from Lodamin-treated or untreatedmice with B16/F10 tumors. Control spleens had large masses compared totreated mice with normal spleen morphology.

FIG. 10C shows the survival curve of treated versus control mice withB16/F10 tumors (n=7). Treatment started at day three (TX) after cellinjection (arrow).

FIG. 11A shows the effect of Lodamin administered orally by gavage onestablished human glioblastoma tumors (U87-MG tumors) growth in nudemice: 15 mg/kg. q.d of Lodmain (solid diamonds) given orally, water(solid squares), (n=5-10 mice per group, *p<0.05).

FIG. 11B shows the effect of Lodamin given in drinking water at a doseof 15 mg/kg q.d. (solid squares), water with Lodamin was changed every 3days. *p<0.05 (results are mean±SE). The mice had established humanglioblastoma tumors (U87-MG tumors) growth.

FIG. 12 shows the Kaplan-Meier survival curve of C57/Bl mice treatedwith Lodamin given at a dose of 15 mg/kg q.d TNP-470 equivalent IP (n=9,white squares), oral (n=10, solid line) or without any treatment (n=10,diamond black).

FIG. 13A shows the effects of Lodamin on angiogenesis in Matrigelimplants, the implants were removed 8 days post injection from mice.

FIG. 13B shows FACS analysis of the Matrigel incorporated cells inLodamin-treated and non-treated mice, CD45-CD31+ represents theendothelial cells.

FIG. 14 shows schematic drawings of diblock-copolymer that is parthydrophilic and part hydrophobic conjugated to MetAP-2 inhibitors anddiblock-copolymer micelles (polymersomes) formed.

FIG. 15 shows schematics of exemplary approaches for functionalizingMetAP-2 inhibitors and coupling copolymers to them.

FIG. 16A shows that Lodamin inhibits angiogenesis and the inflammatoryresponse in the Matrigel plug angiogenesis assay and DTH in mice.Matrigel containing VEGF and bFGF were injected subcutaneously (s.c.,n=5) to determine the effect of Lodamin on angiogenesis and macrophageinfiltration. Upper panel shows representative plugs removed fromLodamin treated or untreated mice (bar=1 cm). Bottom panel shows vesselstaining with anti-CD3 1 antibody and nuclei staining with HematoxilinGill's, bar=100 μm; arrows point to large vessel with open lumans.

FIG. 16B shows FACS dot plots of endothelial cells (CD31+/CD45) ormacrophages (CD45+F4/80+) from Matrigel plugs (marked in the innersquares).

FIG. 16C shows the quantification of infiltrating endothelial cell ormacrophages in Matrigel plugs. Data are presented as a percent ofspecific cell population out of the total cell population (n=3-4,p<0.05).

FIG. 16D shows that Lodamin suppressed ear swelling in DTH reactionselicited by oxazolone, photos of representative ears of Lodamin treatedor untreated mice are circled. Bar=1 cm.

FIG. 16E shows the ear swelling is represented as change in thickness(in μm) compared to original ear thickness. Mice treated with Lodamin(white squares) showed a significant reduction of ear thickness comparedto control mice (black squares). The differences were statisticallysignificant from day 2 to day 12 post challenge, excluding day 5(P<0.05) data are presented as an average±SEM, n=5.

FIG. 16F shows the immunohistological analysis of mice ears post DTHreaction: H&E staining and staining of CD-31 endothelial cell marker.Control ears exhibited an excessive inflammation and edema, includingfibroblast proliferation and spongiosis pointed by arrows. Bar=50 μm.

FIG. 16G shows the representative Matrigel plugs exposed under mouseskin (bar=1 cm). Vehicle treated mice presented bloody plugs surroundedby massive blood vessels, compared to Lodamin treated mice which hadpoor vasculature. Bar=100 μm.

FIG. 16H shows immunohistological analysis of mice ears post DTHreaction: staining of macrophages using F4/80 marker. Control earsexhibited an excessive inflammation. Bar=50 μm. No obvious differencewas found in macrophage distribution, however, the total number ofmacrophages present in control mice was elevated due to greater amountof tissue associated with increased swelling.

FIG. 17A shows the quantification of vessel area (mm²) in differentcorneal assays performed with Lodamin which were administered eitherorally (30 mg/kg q.d), by eye drops (30 mg/ml 1 gtt q.d) or bysubconjunctional injections (30 mg/ml, q.d). Graphs show the significantinhibition of vessel formation after 5 d of treatment. Vessel area wasreduced by 38% after subconjunctional injection (P=0.0002), by 30% andfor eye drops (P=0.003), and by 37% (P=0.04) for oral administration. Ineach group n=10.

FIG. 17B shows representative images taken from the different groups atday 5, bFGF pellet is detected as a white spot in the center of thecornea, blood vessels growing from limbal periphery are reduced inLodamin treated group compared to controls. Vessel area in mm² iscalculated by the following formula: π×clock hours×vessel length(mm)×0.2 mm.

FIG. 18A shows the CNV lesion size of Lodamin-treated mice compared tocontrols. 15 mg/kg/day was given for 7 or 14 d and dose of 30 mg/kg/daywas given for 7 d. CNV lesions in choroidal flat mounts were evaluatedafter staining of blood vessels post blood vessel staining usingisolectin-IB4 conjugated with Alexa® Fluor 488. Data are presented as apercent of blood vessels area in choroidal flat mounts (pixels) oftreatment group out of controls. Data are expressed as mean±SD, (T-test,*P<0.05, **P<0.005, n=10).

FIG. 18B shows H&E stained histological side sections of CNV site inLodamin treated or untreated mice. Substantial differences in fibroustissue thickness and choridal vessel invasion to CNV site are detected,Bars=50 μm.

FIG. 18C shows the FACS analysis of single-cell suspension from retinasafter 3 and 7 d of oral Lodamin treatment. Quantification of macrophageinfiltration in mouse retinas originated from Lodamin treated (30 mg/kg,daily, oral) or mice which were treated with equivalent dose of vehicle.Macrophage population was detected as a double positive CD45+ and F4/80+staining.

FIG. 19A shows the size of CNV lesions after intravitreal injections ofLodamin (100 μg Lodamin, 12.5 μg equivalent to TNP-470 or 300 μg Lodaminequivalent to 37.5 μg TNP-470). Data is presented as mean pixelnumber±SD (n=7-17, *P<0.05, t-test). Lower panels show representativeimages of retinal flat mounts stained with a lectin-FITC, Bars=10 μm.

FIG. 19B shows representative full-field flash ERG responses from theeyes of mice 14 days after intra-ocular injection of vehicle (left) or300 μg/eye Lodamin (right).

FIG. 19C shows representative responses to 8 Hz flicker.

FIG. 19D shows the 5th to 95th prediction limits for ERG parameters invehicle treated rats are shown; the dashed line represents the normalmean of all the parameters. Symbols are parameter values of individualLodamin-treated mice, and represent the same mouse in each column. Dataare ALogNormal (eq. 3). Only the saturating amplitude of the rodphotoresponse (RmP3) differed significantly after Lodamin.

FIG. 19E shows representative images of retinal tissue taken 14 d postintravitreal injection of Lodamin (300 μg/eye) and control naïve mouseretinas. (Bars=50 μm). No apparent retinal tissue changes were detected;both eyes presented normal structures of (1) choroid (2) photoreceptors(3) outer nuclear layer (4) outer plexiform layer (5) inner nuclearlayer (6) inner plexiform layer and (7) ganglion cell layer. The totalretinal thickness remained unchanged.

FIG. 20A shows the levels of VEGF and MCP-1 extracted from whole eyetissues from naïve mice, mice treated with Lodamin (30 mg/kg, oral everyday for 7 d) or Vehicle (same equivalent amount of mPEG-PLA micelles).Data of ELISA assay are presented as a mean of pg/ml concentration±SD(n=6, *P<0.05, t-test).

FIG. 20B shows the ELISA quantification of secreted MCP-1 from RPE tothe medium post 24 h incubation with Lodamin at different doses (0.1-100nM TNP-470 equivalent).

FIG. 20C shows the Western blot protein analysis of samples of tissuelysis of retina or choroid of the different treatment groups: N=naïvemice, V=vehicle treated mice, L=Lodamin treated mice. Factors detectedare TNFα (26 kDa), RAGE-two bands of 35 kDa and ˜50 kDa, HMGB-1 (29kDa), NFidB (detected as two bands ˜65 kDa), MAPK or phosphorylated MAPK(ERK/p-ERK, bands of 42 kDa) and 3-actin (42 kDa). The samples wereanalyzed separately for choroids and retinas. (each sample is originatedfrom 6 eyes).

FIG. 20D is a zymogram showing MMP-2 and MMP-9 activity in ocular tissuefollowing oral Lodamin treatment of mice (30 mg/kg/day 7 d).

FIG. 20E shows the levels of RAGE receptor protein (50 kDa) and itsligand HMGB-1 (29 kDa) in HMVEC cells post TNP-470 treatment atdifferent doses (0.1 nM-100 nM). A dose dependent effect on RAGEexpression was detected.

FIG. 21A shows the quantification of Miles assays performed in miceafter induction of vessel permeability using VEGF (50 ng) and MCP-1 (50pg); extracted dye contents were quantified by measuring at 620 nm(Histograms). Data are expressed as mean±SD (n=10, *P<0.05, t-test).Also shown are representative photos of mouse skin showing diminisheddye in mice treated with oral Lodamin 1 and 3 h before conducting theassay.

FIG. 21B shows that MCP-1 induces vessel permeability in a dosedependent manner. At 10 pg/ml, MCP-1 induced significant vessel leakcompared with PBS.

FIG. 21C shows that Lodamin reduces whole eye vessel permeability asdetermined by Evans blue extraction method. Mouse eyes treated withLodamin (3 h before conducting the assay) were 65% less than thecontrol. Data are presented as mean±SD (n=5, *P<0.05, t-test).

DETAILED DESCRIPTION

The present invention relates in part to compositions and formulationscomprising a MetAP-2 inhibitor associated with a block copolymer,wherein the block copolymer comprises a hydrophobic and hydrophilicmoiety. Described herein are compositions comprising MetAP-2 inhibitorsformulated for oral administration. In some embodiments, the compositioncomprises associating a MetAP-2 inhibitor with a block copolymercomprising a hydrophobic moiety, wherein the hydrophobic moiety is partof a block copolymer comprising a hydrophilic and a hydrophobic moiety.In some embodiments the block copolymer comprising hydrophilic andhydrophobic moiety forms a micelle.

Accordingly, the present invention relates to a method to treat acondition in which MetAP-2 activity is involved in or required for thepathology of the condition, the method comprising administering acomposition comprising a MetAP-2 inhibitor formulated as disclosedherein. In one embodiment, the composition is formulated for oraladministration, although other routes are specifically contemplated. Inone embodiment, the condition is a proliferative condition and/or anangiogenesis-mediated condition, and the method comprises orallyadministering a composition comprising a MetAP-2 inhibitor formulated asdisclosed herein. Other routes of administration are specificallycontemplated, as discussed herein below.

Methionine aminopeptidases are essential for removal of the N-terminalmethionine from nascent proteins. Following this removal, a variety ofmolecules are attached to the unstable, open N-terminus, includingmyristic acid. This acid changes the hydrophobicity of the nascentprotein, and causes specific cellular localization and function. In someinstances, this function may lead to pathological diseases, as with theoncogene Src.⁴⁶ Recently, MetAP-2, one of the 2 major types ofaminopeptidases, has itself been identified as a possible oncogene.⁴⁷Its importance in cell proliferation is well documented.⁴⁵

As a result of this knowledge, numerous classes of therapeutics havebeen synthesized to target and inhibit MetAP-2 activity (including, butnot necessarily limited to over-activity) including the fumagillin andovacillin classes, bengamides, bestatins, pyrimidines, sulfonamides,among others (Bernier 2005). None have clinically succeeded, due partlyto the non-specific nature of their bio-distribution and partly due to apoor PK (pharmacokinetic) profile, such as too rapid clearance, shorthalf life, unacceptable levels of interaction with the nervous system,or crossing the blood-brain barrier. These issues are manifested inunwanted toxic side effects.

Many attempts have been made to overcome these issues associated withboth the reversible and irreversible MetAP-2 classes of inhibitorslargely through small molecule formulation, synthesis or high molecularweight (>60 kDa) polymeric conjugation. However, each formulation hasshown insurmountable short-comings including too-short plasma half-life(a few minutes for the drug and less than 2 hr for the metabolites inthe case of the fumagillin class compound TNP-470), weight loss with thesulfonamides and bestatins, unpredictable cardiovascular events with thebengamides, difficulty in manufacturing, questionable stability, theinability to dose patients over long periods of time, and lack ofclearance of too slow clearance as with higher molecular weight polymerconjugated drugs. Again, the majority of these failures can beattributed to their non-specific biodistribution. It is thereforedesirable to administer a MetAP-2 inhibitor to patients in doses andmethods that are limited in overall toxicities, are stable, and canprovide multiple routes of administration depending on the presentationof the disease.

Further, many MetAP-2 inhibitors, particularly those in the fumagillolclass, are poorly soluble in water (1.9 mg/ml; see U.S. Pat. No.5,536,623), and as such have low absorption and bioavailability if givenorally. Therefore, modification of these MetAP-2 inhibitors is highlydesirable from therapeutic, administration and safety perspectives.

In order to administer a MetAP-2 inhibitor one must overcome severalissues associated with the chemical properties of the small moleculeMetAP-2 inhibitors such as their poor solubility in water, lowabsorption, non-specific bio-distribution and poor bioavailability ifadministered orally, topically, inhaled, or given via eyedrop. Due tothese properties, the vast majority of MetAP-2 inhibitors are currentlyformulated for administration exclusively via injections (i.e.intramuscularly and intravenously).

As demonstrated herein, the inventors have discovered a composition thatformulates MetAP-2 inhibitors such as TNP-470, among others, foralternative routes of administration other than injections, by bindingthe MetAP-2 inhibitor to a diblock copolymer, for example a diblockco-polymer comprising PEG-PLA. The inventors have discovered that suchconjugation of a MetAP-2 inhibitor with block copolymers (hereinreferred to as “block copolymer-MetAP-2 inhibitor conjugates”) is ahighly suitable formulation for oral, topical, inhaled, eyedrop orperitoneal administration. While having advantages for oral or othernon-injected routes of administration, is also contemplated herein thatthe block copolymer compositions described herein can also beadministered by injection (e.g., intramuscular, subcutaneous, etc.).

The inventors demonstrate that a MetAP-2 inhibitor was successfullyconjugated to a modified PEG-PLA polymer through its amine, and formednano-size polymersomes. The inventors also demonstrate, using imagestaken by TEM, that spherical micelles were formed, and size measurementwith DLS showed a low range of size distribution around 10 nanometers.By using confocal microscopy images of fluorescently—labeledpolymersomes, the inventors demonstrate rapid uptake by Human UmbilicalVein Endothelial Cells (HUVECs), and when the MetAP-2 inhibitorpolymersomes were added, a significant inhibition of HUVEC proliferationwas shown (as compared to no effect of the carrier itself).

In-vivo studies done on mice showed a significant inhibition ofsubcutaneous Lewis Lung Carcinoma tumors with C57/BL mice given a dailyoral administration of TNP470 micelles. A dose of 15 mg/kg TNP-470equivalent showed 63% inhibition without any weight loss to the mice.

Accordingly, the inventors have discovered that conjugating a MetAP-2inhibitor such as TNP-470 to diblock copolymers is useful forformulation as an oral administration formulation and that such aMetAP-2 inhibitor-diblock copolymer conjugate is useful in a method totreat conditions that involve or require MetAP-2 activity for theirpathologies, such as cancer, RA, viral infection, bacterial or fungalinfection among others, and those associated with abnormal orhyperpermeable vasculature such as edema, age-related maculardegeneration, diabetic retinopathy, inflammation or other conditionsinvolving or requiring MetAP-2 activity as described herein or as knownin the art.

One embodiment, for the first time, is an oralanti-prolierative/anti-angiogenic MetAP-2 inhibitor polymeric drug withpotent anti-tumor and anti-metastatic efficacy named Lodamin. Theinventors characterized its physicochemical properties and show thatthis polymeric pro-drug derived from TNP 470 successfully overcomes thedrug limitations while retaining its anti-proliferative andantiangiogenic activities. Lodamin is produced by covalent conjugationof TNP 470 to a di-block copolymer: PEG PLA. The amphiphilic nature ofthis polymeric drug enables self-assembly of micelles in an aqueousmedium²⁶. In this structure, the TNP-470 is located in the core, whereit is protected from the acidic environment of the stomach, thusenabling oral availability. Furthermore, advantage is taken of usingbiocompatible and well characterized polymers^(27, 28).

Lodamin, administered orally, is effectively absorbed in the intestineand accumulates in tumor tissue. The drug significantly inhibits cellproliferation and angiogenesis, as demonstrated by inhibition of HUVECproliferation, in the corneal micropocket assay, and in mouse tumormodels. Lodamin significantly inhibited primary tumor growth asdemonstrated in models of melanoma and lung cancer. Notably, oralLodamin successfully prevented liver metastasis of melanoma tumor cellswithout causing liver-toxicity or other side-effects and led toprolonged mouse survival.

Moreover, unlike the neurological impairments caused by free TNP-470,Lodamin does not penetrate the blood-brain barrier, and accordingly didnot cause neurotoxicity in mice. These results indicate that Lodamin isa good candidate for a safe maintenance drug with effective anti-tumorand anti-metastatic properties.

Accordingly, the inventors have discovered that conjugating MetAP-2inhibitors to diblock copolymers is useful for formulation such as anoral administration formulation, among others, and that such afumagillol derivative-diblock copolymer conjugate is useful in a methodto treat proliferative disorders, including angiogenesis-mediatedconditions and disorders such as cancer and AMD, among many others.

The inventors also showed that Lodamin, a polymeric conjugate ofTNP-470, is a potent broad spectrum antiangiogenic ophthalmic drug.Lodamin significantly reduced angiogenesis, vascular leakage andinflammation in the murine models of choroidal neovascularization (CNV)and corneal neovascularization. Lodamin was found to suppress angiogenicand inflammatory signals including monocyte chemotactic protein-1(MCP-1). Additionally, Lodamin reduced levels of the receptor foradvanced glycation end products (RAGE) which likely contributes to itsbroad spectrum of activity (see Example 7, FIGS. 16-21).

Accordingly, described herein is a method of treating a conditioninvolving or relying upon MetAP-2 activity for its pathology, the methodcomprising administering a MetAP-2 inhibitor formulation comprising aMetAP-2 inhibitor having anti-proliferative activity, covalently linkedto a block copolymer comprising a hydrophilic polymer moiety and ahydrophobic polymer moiety. In one embodiment, the condition is selectedfrom the group consisting of cancer, metastatic tumors, psoriasis,age-related macular degeneration (AMD), thyroid hyperplasia,preeclampsia, rheumatoid arthritis and osteo-arthritis, Alzheimer'sdisease, obesity, pleural effusion, atherosclerosis, endometriosis,diabetic/other retinopathies, ocular neovascularizations, IL-2 therapyassociated edema and other edemas, malaria, SARS, HIV, herpes, lupus,IPF, COPD, asthma, cystic fibrosis, transplant rejection, allergicreaction, multiple sclerosis, bacterial infection, viral infection,conditions involving or characterized by vascular hyperpermeability,inflammation, and spinal injury.

In one embodiment, described herein is a method of treating anangiogenesis-mediated condition, the method comprising orallyadministering a composition comprising a formulation of a fumagillolderivative that retains anti-angiogenic activity, the formulationcomprising the fumagillol derivative is associated with a blockcopolymer comprising a hydrophilic polymer moiety and a hydrophobicpolymer moiety. In one embodiment, the angiogenesis-mediated conditionis selected from a group comprising cancer, metastatic tumors,psoriasis, age-related macular degeneration (AMD), thyroid hyperplasia,preclampsia, rheumatoid arthritis and osteo-arthritis, Alzheimer'sdisease, obesity, pleural effusion, atherosclerosis, endometriosis,diabetic/other retinopathies, ocular neovascularizations, IL-2 therapyassociated edema and other edemas.

In one embodiment, described herein is a method of treating ocularneovascularization in a subject in need thereof, the method comprisingadministering a MetAP-2 inhibitor formulation comprising a MetAP-2inhibitor having anti-proliferative activity, covalently linked to ablock copolymer comprising a hydrophilic polymer moiety and ahydrophobic polymer moiety. In one embodiment, the MetAP-2 inhibitor isa fumagillol derivative. In some aspects, the MetAP-2 inhibitorformulation described herein can be applied to an eye disease ordisorder which involves neovascularization, and/or edema.

In another embodiment, provided herein is a method of treating ocularneovascularization in a subject in need thereof, the method comprisingadministering a composition comprising a formulation of a fumagillolderivative that retains anti-angiogenic activity, wherein theformulation comprising the fumagillol derivative is associated with ablock copolymer comprising a hydrophilic polymer moiety and ahydrophobic polymer moiety. Examples of ocular neovascularizationdiseases or disorders include but are not limited to AMD, ARMD,proliferative diabetic retinopathy (PDR), and retinopathy of prematurity(ROP).

In one embodiment, provided herein is a composition comprising amethionine aminopeptidase-2 (MetAP-2) inhibitor formulation havinganti-proliferative activity, wherein the formulation comprises a MetAP-2inhibitor covalently linked to a block copolymer comprising ahydrophilic polymer moiety and a hydrophobic polymer moiety.

In another embodiment, provided herein is a method of making a diblockcopolymer composition comprising a MetAP-2 inhibitor that hasanti-proliferative activity, the method comprising conjugating theMetAP-2 inhibitor to a diblock copolymer comprising a hydrophilicpolymer moiety and a hydrophobic polymer moiety.

In another embodiment, provided herein is a diblock copolymercomposition comprising a MetAP-2 inhibitor, the composition produced bythe methods described herein.

In another embodiment, provided herein is a composition comprising aformulation of a fumagillol derivative that has anti-angiogenicactivity, the formulation comprising the derivative associated with ablock copolymer comprising a hydrophilic polymer moiety and ahydrophobic polymer moiety.

In one embodiment, provided herein is a method of making a diblockcopolymer micelle comprising a fumagillol derivative that hasanti-angiogenic activity, the method comprising conjugating thefumagillol derivative to a diblock copolymer comprising a hydrophilicpolymer moiety and a hydrophobic polymer moiety and forming micelles ofthe resulting conjugate.

In one embodiment of all aspects described herein, MetAP-2 inhibitor isan irreversible MetAP-2 inhibitor.

In one embodiment of all aspects described herein, MetAP-2 inhibitor isa reversible MetAP-2 inhibitor.

In one embodiment of this and all other aspects described herein, theblock copolymer is a diblock copolymer.

In one embodiment of this and all other aspects described herein, theformulation comprises a micelle comprising the block copolymerassociated with the fumagillol derivative.

In one embodiment of all aspects described herein, the fumagillolderivative thereof is associated with the hydrophobic moiety of theblock copolymer.

In one embodiment of all aspects described herein, the hydrophobicpolymer moiety of the block copolymer is selected from the groupconsisting of poly(d,L-lactic acid), poly(L-lysine), poly(asparticacid), poly(caprolactone) (PCL), and poly(propylene oxide).

In one embodiment of all aspects described herein, the hydrophobicmoiety is a poly(d,L-lactic acid) (PLA) polymer.

In one embodiment of all aspects described herein, MetAP-2 inhibitor isassociated with the PLA moiety of the diblock copolymer.

In one embodiment of this and all other aspects described herein, thehydrophilic polymer moiety of the block copolymer is polyethylene glycol(PEG). In one embodiment of all aspects described herein, the PEGpolymer is a capped PEG polymer.

In one embodiment of this and all other aspects described herein, theblock copolymer is a diblock copolymer comprising a PEG-PLA diblockcopolymer having hydrophilic PEG and hydrophobic PLA moieties.

In one embodiment of this and all other aspects described herein, theanti-angiogenic activity is an anti-tumor activity.

In one embodiment of this and all other aspects described herein, thefumagillol derivative comprises a derivative selected from the groupconsisting of 6-O—(N-chloroacetylcarbamoyl) fumagillol (TNP-470),6-O-(4-methoxyaniline)acetyl fumagillol;6-O-(3,4,5-trimethexyaniline)acetyl fumagillol;6-O-(4-(N,N-dimethylethoxy) aniline)acetyl fumagillol;6-O-(cyclopropylamino)acetyl fumagillol; 6-O-(cyclobutylamino)acetylfumagillol; 4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy-1,5 20dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol; and4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol.

In one embodiment of this and all other aspects described herein, thefumagillol derivative comprises 6-O—(N-chloroacetylcarbamoyl)fumagillol(TNP-470).

In one embodiment of this and all other aspects described herein, theformulation comprises a diblock copolymer micelle formed with thediblock copolymer described herein.

In one embodiment of this and all other aspects described herein, thefumagillol derivative is associated with the PLA moiety of the diblockcopolymer.

In one embodiment of this and all other aspects described herein, thecomposition or formulation is administered orally.

In one embodiment of this and all other aspects described herein, thecomposition or formulation is administered by intravitreous injection.In other embodiments, the composition or formulation is for topicaladministration, IV administration, peritoneal administration, injection,ocular administration, suppository administration, pulmonaryadministration or inhalation, and nasal administration.

In one embodiment, the composition is administered by intravitreousinjection.

Definitions

The term “MetAP-2 inhibitor” refers to an agent that, at a minimum,inhibits the activity of MetAP-2 by at least 20% in a MetAP-2 assay asdescribed in U.S. Pat. No. 6,548,477 or in U.S. Pat. No. 7,030,262(which are both incorporated herein by reference), preferably at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or more, upto and including complete or 100% inhibition relative to the absence ofsuch an agent. Reversible and irreversible MetAP-2 inhibitors are knownin the art and are encompassed by the term “MetAP-2 inhibitor.”Fumagillol derivatives as described herein that have anti-proliferativeactivity are MetAP-2 inhibitors as the term is used herein. Thefumagillol and ovacilin classes of MetAP-2 inhibitors have a reactivespiroepoxide moiety that reacts to form a covalent bond with theimidazole nitrogen of histidine 231 in the catalytic site of MetAP-2.Molecular modeling with potential irreversible MetAP-2 inhibitors willpermit the skilled artisan to readily predict whether a given candidateinhibitor will react with histidine 231 of the enzyme and therefore havethe desired inhibitory activity. Similarly, a rational design approachcan be used to prepare reversible MetAP-2 inhibitors, as demonstrated byWang et al.⁴³

The term “fumagillol derivative” encompasses compounds represented bythe Formula I below:

wherein, X is —OH and Y is halogen, or X and Y are linked together toform a oxyrane ring, B represents —(C═O)— or —CH2-, R1 representshydrogen, hydroxyl, —CN; NO2, —CF3; formyl; C₁-C₄ thioalkyl, acetamido;acetoxy; C₁-C₆ alkyl, C₁-C₄ aminoalkyl, C₁-C₄ alkylaminoalkyl; C₁-C₄dialkylaminoalkyl; C₁-C₆ alkyloxy, C₁-C₆ aminoalkyloxy; C₁-C₄alkylaminoalkoyy, C₁-C₄ dialkylaminoalkoxy, amino; C₁-C₆ alkylamino;C₁-C₄ dialkylamino; C1-C4 hyrdoalkyl; C₁-C₄ alkyloxycarboxylic acid etc.Fumagillol derivatives are disclosed in European Patent Application0354787, 0357061, and 0415294 and Japanese patent applicationJP-A01-233275 and U.S. Pat. No. 5,290,807, which are incorporated hereinin their entirety by reference. Other MetAP-2 inhibitory fumagillolderivatives, such as PPI-2458, are described by Bernier et al., Proc.Natl. Acad. Sci. U.S.A. 101: 10768-10773 (2004). Fumagillol derivativesas described herein have, at a minimum, anti-angiogenic activity whentested, for example, in a HUVEC proliferation assay as known in the artand described herein below. It should be understood that the term“fumagillol derivative” refers to the derivatives of fumagillol ofFormula I, whereas a “fumagillol derivative block copolymer conjugate”refers to such derivatives conjugated to a block copolymer. Similarly, a“MetAP-2 inhibitor” refers to the inhibitor itself, while a “MetAP-2inhibitor block copolymer conjugate” refers to the MetAP-2 inhibitorconjugated to a block copolymer.

As used herein, the term “proliferation” refers to the development ofcells that result in unwanted or undesirable physiological consequences,such as with a tumor or inflammation or hyperpermeable, abnormalvasculature, viral infections, bacterial infections and fungalinfections. Angiogenesis should be understood to be a proliferativeprocess. Thus, in certain instances, the term “proliferation” can applyto the development of blood vessels. Such development is also referredto herein as “angiogenesis.” In some instances, the term “angiogenesis”,as used herein refers to the sprouting of new blood vessels frompre-existing blood vessels, characterized by endothelial cellproliferation and migration triggered by certain pathologicalconditions, such as the growth of tumors, metastasis, AMD and arthritis,among others. It should be noted that the term “proliferation” can alsoapply to the proliferation of viruses, bacteria, fungi, microsporidia,etc. The context of the term will make it clear which type of“proliferation” is being referred to.

The term “anti-proliferative activity” refers to the property of anagent that inhibits, suppresses or reduces the rate of growth orcreation of new, undesired cells or blood vessels in the body in orderto combat disease. A compound or agent with anti-proliferative activityas used herein is an agent that inhibits MetAP-2 and leads to thesuppression of unwanted proliferating cells, e.g., in unwantedangiogenesis. Also encompassed by the term “anti-proliferative activity”is an agent that inhibits MetAP-2 and leads to cell death.

The term “anti-angiogenesis activity” as used herein refers to an agentwhich inhibits or suppresses or reduces the rate of growth or creationof new blood vessels in the body in order to combat disease. A compoundor agent with anti-angiogenesis activity as used herein is an agentcapable of inhibiting the formation of blood vessels or the formation ofvasculature with abnormal or hyperpermeable properties. A diseaseassociated with vascular permeability includes vascular complications ofdiabetes such as non-proliferative diabetic retinopathy and diabeticnephropathy, nephrotic syndrome, pulmonary hypertension or fibrosis,burn edema, tumor edema, brain tumor edema, IL-2 therapy-associatededema, and other edema-associated diseases, inflammatory disorders,complications from spinal injury, fibrotic disorders, and infections,including viral infections, bacterial infections and fungal infections.

As used herein, the term “retains anti-proliferative activity” meansthat a given compound derived from a MetAP-2 inhibitor has at least 20%of the anti-proliferative activity of that MetAP-2 inhibitor. Thus, agiven fumagillol derivative will “retain anti-proliferative activity” ifit retains at least 20% of the anti-proliferative activity offumagillol.

As used herein, the term “retains anti-angiogenesis activity” means thata given fumagillol derivative has at least 20% of the anti-angiogenicactivity of TNP-470 in a HUVEC assay of angiogenesis as describedherein.

MetAP-2 inhibitor formulations as described herein can also be used toprevent the leakage of cell proliferation stimulators from bloodvessels. That is, MetAP-2 inhibitors can reduce or prevent vascularhyperpermeability, and prevent the accumulation of stimulatory factorsat sites of such permeability.

As used herein, the term “inhibit” or “inhibition” means the reductionor prevention of tumor growth and/or tumor metastasis in cancers.Inhibition includes slowing the rate of tumor growth and metastasis. Thetumor growth rate can be reduced by about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about125%, about 150% or more compared to a control, untreated tumor of thesame type Inhibition also means a reduction in the size of the tumor(including, but not limited to a reduction caused by cytotoxic activity)of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, up to andincluding 100% (no tumor) when compared to a control, untreated tumor ofthe same type. The prevention of tumor growth and/or metastasis means nofurther increase in the size of the tumors from the time of start oftreatment administration. Prevention also means status quo of no newmetastatic tumors detected (ie. no further spread of cancer) and/or noincreased amount of tumor markers detected by methods known in the art.

As used herein, the term “MetAP-2-mediated condition” refers to diseasesor disorders in which MetAP-2 activity contributes to the pathology ofthe condition, whether it involves inappropriate cell proliferation,e.g., inappropriate angiogenesis, or, for example, vascularhyperpermeability resulting from the MetAP-2 activity. While MetAP-2over-expression or over-activity can be involved, the expression oractivity involved in a condition is not necessarily over-expression orover-activity—that is, normal levels of MetAP-2 activity can alsocontribute to pathological conditions and should be considered a targetfor therapy in such instances. Examples of conditions in which MetAP-2activity is involved in or required for the pathology include, but arenot limited to cancer, metastatic tumors, psoriasis, age-related maculardegeneration (AMD), thyroid hyperplasia, preeclampsia, rheumatoidarthritis and osteo-arthritis, Alzheimer's disease, obesity, pleuraleffusion, atherosclerosis, endometriosis, diabetic/other retinopathies,ocular neovascularizations, IL-2 therapy associated edema and otheredemas, malaria, SARS, HIV, herpes, lupus, IPF, COPD, asthma, cysticfibrosis, transplant rejection, allergic reaction, multiple sclerosis,bacterial infection, viral infection, and conditions involving orcharacterized by vascular hyperpermeability. Angiogenesis-mediatedconditions are a sub-set of MetAP-2-mediated conditions, diseases ordisorders that are the direct result of aberrant blood vesselproliferation (e.g. diabetic retinopathy and hemangiomas, among othersknown in the art and/or discussed herein).

As used herein, the term “angiogenesis-mediated condition” refers todiseases or disorders that are dependent on a rich blood supply andblood vessel proliferation for the disease pathological progression (eg.metastatic tumors) or diseases or disorders that are the direct resultof aberrant blood vessel proliferation (e.g. diabetic retinopathy andhemangiomas). Non-limiting examples include abnormal vascularproliferation, neovascularization, hyperpermeability, ascites formation,psoriasis, age-related macular degeneration, retinopathy, thyroidhyperplasia, preeclampsia, rheumatoid arthritis and osteo-arthritis,Alzheimer's disease, obesity, pleural effusion, atherosclerosis,endometriosis, diabetic/other retinopathies, ocular neovascularizationssuch as neovascular glaucoma and corneal neovascularization.

As used herein, the term “tumor” means a mass of transformed cells thatare characterized, at least in part, by containing angiogenicvasculature. The transformed cells are characterized by neoplasticuncontrolled cell multiplication which is rapid and continues even afterthe stimuli that initiated the new growth has ceased. The term “tumor”is used broadly to include the tumor parenchymal cells as well as thesupporting stroma, including the angiogenic blood vessels thatinfiltrate the tumor parenchymal cell mass. Although a tumor generallyis a malignant tumor, i.e., a cancer having the ability to metastasize(i.e. a metastatic tumor), a tumor also can be nonmalignant (i.e.non-metastatic tumor). Tumors are hallmarks of cancer, a neoplasticdisease the natural course of which is fatal. Cancer cells exhibit theproperties of invasion and metastasis and are highly anaplastic. Tumorsare among the angiogenesis-mediated diseases encompassed by thetherapeutic methods described herein.

As used herein, the term “tumor” is also used in reference to specifictypes of tumors, e.g., brain tumors including neuroblastoma,medulloblastoma, meningioma and glioblastoma; head and neck cancer,thyroid carcinoma, endocrine tumors, esophageal cancer, small cell andnon-small cell lung cancer, colon cancer, rectal cancer, pancreaticcancer, gastric cancer, bladder cancer, hepatic cancer, malignantlymphoma, acute and chronic leukemia, Kaposi's sarcoma, glioma,hemangioma, osteosarcoma, soft tissue sarcoma, malignant melanoma, skincancer, prostate cancer, breast carcinoma, choriocarcinoma, ovariancancer, cervical cancer, uterine cancer and mesenchymal tumors, amongothers. In the context of the methods and compositions disclosed herein,and as highlighted by the inclusion of lymphomas and leukemias on thelist above, it should also be understood that non-solid “tumors” canalso benefit from the administration of MetAP-2 inhibitors formulated asdescribed herein.

As used herein, the term “metastases” or “metastatic tumor” refers to asecondary tumor that grows separately elsewhere in the body from theprimary tumor and has arisen from detached, transported cells, whereinthe primary tumor is a solid tumor. The primary tumor, as used herein,refers to a tumor that originated in the location or organ in which itis present and did not metastasize to that location from anotherlocation. As used herein, a “malignant tumor” is one having theproperties of invasion and metastasis and generally showing a highdegree of anaplasia. Anaplasia is the reversion of cells to an immatureor a less differentiated form, and it occurs in most malignant tumors.

The terms “polymersomes” and “polymeric micelles” are usedinterchangeably herein to refer to the same block copolymercompositions.

The term “copolymer” also known as “heteropolymer” as used herein refersto a polymer derived from two (or more) monomeric species, as opposed toa homopolymer where only one monomer is used. Copolymerization refers tomethods used to chemically synthesize a copolymer.

The term “block copolymer” as used herein refers to the polymercomprising more than one subunit (or oligomer) type, wherein thecopolymer comprises regions of a polymer comprising one subunit typeadjoined to a polymer region comprising a second subunit type, forexample the term block copolymer refers to a copolymer comprised of twoor more homopolymer subunits linked by covalent bonds. The union of thehomopolymer subunits may require an intermediate non-repeating subunit,known as a junction block. Block copolymers are made up of blocks ofdifferent polymerized monomers. Block copolymers are interesting becausethey can “microphase separate” to form periodic nanostructures. Blockcopolymers are described in further detail in the section “Copolymers”herein below.

The term “diblock copolymer” as used herein refers to a block copolymerwith two distinct blocks. A block copolymer with three distinct blocksis called a triblock copolymers. It is also possible to havetetrablocks, multiblocks, etc.

The term “hydrophilic” as used herein refers to a molecule or portion ofa molecule that is typically charge-polarized and capable of hydrogenbonding, enabling it to dissolve more readily in water than in oil orother hydrophobic solvents. Hydrophilic molecules are also known aspolar molecules and are molecules that readily absorb moisture, arehygroscopic, and have strong polar groups that readily interact withwater. A “hydrophilic” polymer as the term is used herein, has asolubility in water of at least 100 mg/ml at 25° C.

The term “hydrophobic” as used herein refers molecules tend to benon-polar and prefer other neutral molecules and non-polar solvents.Hydrophobic molecules in water often cluster together. Water onhydrophobic surfaces will exhibit a high contact angle. Examples ofhydrophobic molecules include the alkanes, oils, fats, and greasysubstances in general. Hydrophobic materials are used for oil removalfrom water, the management of oil spills, and chemical separationprocesses to remove non-polar from polar compounds. Hydrophobicmolecules are also known as non-polar molecules. Hydrophobic moleculesdo not readily absorb water or are adversely affected by water, e.g., asa hydrophobic colloid. A “hydrophobic” polymer as the term is usedherein has a solubility in water less than 10 mg/ml at 25° C.,preferably less than 5 mg/ml, less than 1 mg/ml or lower.

The term “hydrophobic drug” as used herein refers to any organic orinorganic compound or substance having biological or pharmacologicalactivity and adapted or used for a therapeutic purpose having a watersolubility less than 10 mg/ml. MetAP-2 inhibitors, in particularfumagillol derivatives, tend to be hydrophobic drugs.

The term “micelle” as used herein refers to an arrangement of surfactantmolecules (surfactants comprise a non-polar, lipophilic “tail” and apolar, hydrophilic “head”). As the term is used herein, a micelle hasthe arrangement in aqueous solution in which the non-polar tails faceinward and the polar heads face outward. Micelles are typically colloidparticles formed by an aggregation of small molecules and are usuallymicroscopic particles suspended in some sort of liquid medium, e.g.,water, and are between one nanometer and one micrometer in size. Atypical micelle in aqueous solution forms an aggregate with thehydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic tail regions in the micelle center. Thistype of micelle is known as a normal phase micelle (oil-in-watermicelle). Inverse micelles have the headgroups at the centre with thetails extending out (water-in-oil micelle). Micelles are approximatelyspherical in shape. Other phases, including shapes such as ellipsoids,cylinders, and bilayers are also possible. The shape and size of amicelle is a function of the molecular geometry of its surfactantmolecules and solution conditions such as surfactant concentration,temperature, pH, and ionic strength. The process of forming micellae isknown as micellization.

The term “therapeutically effective amount” refers to an amount that issufficient to effect a therapeutically or prophylactically significantreduction or measurable suppression of a marker or symptom associatedwith a disease, condition or disorder dependent upon MetAP-2 activityfor its pathology when that amount is administered to a typical subjectwho has such a condition. A therapeutically or prophylaticallysignificant reduction in a marker or symptom is, e.g. about 10%, about20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,about 90%, up to and including 100%, i.e., no symptoms or a marker atlevels characteristic of non-diseased individuals, as compared to acontrol or non-treated subject. Alternatively, or in addition, a“therapeutically effective amount” can refer to an amount which directlyor indirectly provides a reduction (i.e., at least a 10% reduction,preferably at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95% ormore) in the expression or activity of MetAP-2. In some embodimentswhere the condition is, for example, cancer, the term “therapeuticallyeffective amount” refers to the amount that is safe and sufficient toprevent or delay the development and further spread of metastases incancer patients. The amount can also cure or cause the cancer to go intoremission, slow the course of cancer progression, slow or inhibit tumorgrowth, slow or inhibit tumor metastasis, slow or inhibit theestablishment of secondary tumors at metastatic sites, or inhibit theformation of new tumor metastasis.

The term “treat” or “treatment” refer to both therapeutic treatment andprophylactic or preventative measures (prevention is also understood torefer to a reduction in the likelihood of developing disease, e.g., atleast a 20% reduced likelihood), wherein the object is to prevent orslow down the development or spread of disease. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadydiagnosed with disease. Where the disease is, e.g., cancer, those inneed of treatment include those likely to develop metastases.

The terms “composition” or “pharmaceutical composition” are usedinterchangeably herein and refers to compositions or formulations thatusually comprise an excipient, such as a pharmaceutically acceptablecarrier that is conventional in the art and that is suitable foradministration to mammals, and preferably humans or human cells. Suchcompositions can be specifically formulated for administration via oneor more of a number of routes, including but not limited to oral, IV,peritoneal, injected (e.g., subcutaneous, intramuscular, etc.), eyedropor ocular, suppository, topical, pulmonary, including inhaled, and nasalroutes, among others.

The term “polymeric drug delivery composition” as used herein refers tothe combination of drug, and block copolymer.

As used herein, the term “medicament” refers to an agent that promotesthe recovery from and/or alleviates a symptom of a relevant condition.

The “pharmaceutically acceptable carrier” means a pharmaceuticallyacceptable means to mix and/or deliver the targeted delivery compositionto a subject. The term “pharmaceutically acceptable carrier” as usedherein means a pharmaceutically acceptable material, composition orvehicle, such as a liquid or solid filler, diluent, excipient, solventor encapsulating material which solubilizes, stabilizes or otherwiseprovides a drug formulation with properties necessary to deliver thedrug to an individual in a controlled format. A “carrier” can also beinvolved in carrying or transporting the subject agents from one organ,or portion of the body, to another organ, or portion of the body. Eachcarrier must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and is compatible withadministration to a subject, for example a human. A diblock copolymer asdescribed herein is a pharmaceutically acceptable carrier as the term isused herein. Other pharmaceutically acceptable carriers can be used incombination with the block copolymer carriers as described herein. Apharmaceutically acceptable carrier does not promote the raising of animmune response to the drug.

The terms “polymer solution,” “aqueous solution” and the like, when usedin reference to a block copolymer contained in such a solution, refer towater, i.e. aqueous, based composition having such block copolymer, orparticularly to copolymer conjugates as described herein, such as aMetAP-2 inhibitor-PLA-PEG conjugate dissolved therein at a functionalconcentration. Polymer solution includes all free flowing forms of thecomposition comprising the conjugated copolymers as described herein andwater. Polymer solutions act to solubilize the drug in a form that isacceptable for administration at physiologically relevant temperatures(temperatures <45° C.).

The term “aqueous solution” as used herein includes water withoutadditives, or aqueous solutions containing additives or excipients suchas pH buffers, components for tonicity adjustment, antioxidants,preservatives, drug stabilizers, etc., as commonly used in thepreparation of pharmaceutical formulations.

The term “drug formulation” as used herein refers to all combinations ofdrug with polymer, for example polymer solutions that are mixed withdrug to form drug solutions, as well as mixtures of undissolved polymerwith drug, i.e. polymeric drug delivery compositions, that aresubsequently dissolved into an aqueous environment to form a drugsolution.

The term “administration” as used herein refers to the presentation offormulations to humans and animals in effective amounts, and includesall routes for dosing or administering drugs, whether self-administeredor administered by medical practitioners. Oral administration ispreferred.

The term “biodegradable” as used herein means the block copolymer canchemically break down or degrade within the body to form nontoxiccomponents. The rate of degradation can be the same or different fromthe rate of drug release.

The term “PLA” as used herein refers to a polymer derived from thecondensation of lactic acid or by the ring opening polymerization oflactide.

The term “biodegradable polyesters” as used herein refers to anybiodegradable polyesters, which are preferably synthesized from monomersselected from the group consisting of D,L-lactide, D-lactide, L-lactide,D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolicacid,ε-caprolactone, ε-hydroxy hexanoic acid, γ-butyrolactone, γ-hydroxybutyric acid, δ-valerolactone, δ-hydroxy valeric acid, hydroxybutyricacids, malic acid, and copolymers thereof.

As used herein, the term “patient” refers to a mammal, including ahuman, in need of the treatment to be administered.

The term “subject” and “individual” are used interchangeably herein, andrefer to an animal, for example a human, to whom treatment, includingprophylactic treatment, with a composition as described herein, isprovided. The term “mammal” is intended to encompass a singular “mammal”and plural “mammals,” and includes, but is not limited: to humans,primates such as apes, monkeys, orangutans, and chimpanzees; canids suchas dogs and wolves; felids such as cats, lions, and tigers; equids suchas horses, donkeys, and zebras, food animals such as cows, pigs, andsheep; ungulates such as deer and giraffes; rodents such as mice, rats,hamsters and guinea pigs; and bears. Preferably, the mammal is a humansubject. As used herein, a “subject” refers to a mammal, preferably ahuman. The term “individual”, “subject”, and “patient” are usedinterchangeably

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural references unless the context clearlydictates otherwise. Thus, for example, reference to a composition fordelivering “a drug” includes reference to two or more drugs. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

MetAP-2 Inhibitors

There are a number of MetAP-2 inhibitors known in the art, and MetAP-2inhibitors of any kind are contemplated for formulation and use asdescribed herein. That is, it is specifically contemplated herein thatany of them can beneficially be prepared as block copolymer conjugatesfor delivery to a subject to treat a disease, disorder or conditionrelated to, associated with or requiring MetAP-2 activity for itspathology. MetAP-2 inhibitors formulated as described herein retainanti-proliferative activity—that is, a MetAP-2 inhibitor formulated witha block copolymer conjugate as described herein will retain a meaningfulportion of the anti-proliferative (including, but not limited toanti-angiogenic) activity of the inhibitor alone as measured in aMetAP-2 assay as described herein.

More specifically, however, MetAP-2 inhibitors including, but notlimited to fumagillol derivatives, ovalicin, anthranilic acidsulfonamides (Wang et al., Proc. Natl. Acad. Sci. U.S.A. 105: 1838-1843(2008)) e.g., A-800141, bengamides, bestatins (including, but notlimited to reversible MetAP-2 inhibitors such as A-357300 and othersdescribed by Wang et al., Cancer Res. 63: 7861-7869), triazoles (see,e.g., compounds described in U.S. Pat. No. 7,303,082, and compoundsdescribed, e.g., by Garrabrant et al., Angiogenesis 7: 91-96 (2004)),3-amino-2-hydroxyamides, hydroxyamides and acylhydrazines arecontemplated for use in the formulations and methods described herein.

MetAP-2 inhibitors of various classes are described, for example, inU.S. Pat. Nos. 7,348,307, 7,268,111, 7,157,420, 7,105,482, 7,084,108,7,037,890, 6,919,307, 6,548,477, 6,242,494, 6,288,228, 6,849,757,6,887,863, 4,831,135, 7,122,345 and 7,030,262 and in U.S. publishedPatent Applications 20070254843, 20070161570, 20070117758, 20070010452,20060223758, 20060069028, 20050239878, 20050059585, 20030109671,20020193298, and 20020151493, the disclosures of each of which areincorporated herein by reference. Each of the MetAP-2 inhibitorsdescribed is potentially suitable for formulation as a MetAP-2 inhibitorblock copolymer conjugate as described herein, for the treatment ofMetAP-2 associated diseases, disorders or conditions.

Standard chemical approaches known to those of skill in the art can beused to conjugate a MetAP-2 inhibitor to block copolymer to generate acomposition as described herein; the specific approach will depend uponthe type of MetAP-2 inhibitor selected. For example, MetAP-2 inhibitorswith alcohol functionality can be coupled to PEG-PLA using the same typeof synthesis used to functionalize fumagillol (e.g., reaction withchloroacetylisocyanate, followed by reaction with a polymer bearingprimary amine functionality). As an example, the compound A-357300 canbe coupled in this manner after amine protection. See, e.g., FIG. 15.Similar coupling for can also be used, for example, to couple compoundssuch as (1-hydroxymethyl-2-methyl-propyl)-carbamicacid-(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-ylester (see U.S. Pat. No. 6,548,477). Additional fumagillin derivativeswith alcohol functionalities permitting such conjugation are described,for example, in U.S. Pat. No. 7,087,768 (Han et al.)—see, e.g., Example35 therein.

Assays for MetAP-2 activity are also known in the art. Assays aredescribed, for example in U.S. Pat. Nos. 6,548,477 and 7,030,262. Anysuch assay can be used to evaluate the MetAP-2 inhibitory activity of agiven compound or the activity of a given MetAP-2 inhibitor blockcopolymer conjugate as described herein. For the avoidance of doubt,however, a MetAP-2 inhibitor will demonstrate MetAP-2 inhibition (by atleast 20%, preferably by at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, up to and including 100%(complete inhibition)) at a dosage or concentration that issubstantially not toxic to the cells or subject to which it is to beadministered, in either of the following two MetAP-2 assays.

Recombinant human MetAP-2 is expressed and purified from insect cells asdescribed in Li and Chang, (1996) Biochem. Biophys. Res. Commun.227:152-159. Various amounts of candidate MetAP-2 inhibitor compound arethen added to buffer H (10 mM Hepes, pH 7.35, 100 mM KC 1, 10% glycerol,and 0.1 M Co.sup.2+) containing 1 nM purified recombinant human MetAP-2and incubated at 37° C. for 30 minutes. To start the enzymatic reaction,a peptide containing a methionine residue, e.g., Met-Gly-Met, is addedto the reaction mixture to a concentration of 1 mM. Released methionineis subsequently quantified at different time points (e.g., at 0, 2, 3,and 5 minutes) using the method of Zou et al. (1995) Mol. Gen Genetics246:247-253). MetAP-2 inhibition is scored relative to a control lackingthe candidate inhibitor, and can also be scored relative to a knownMetAP-2 inhibitor, e.g. TNP-470 or, for example, TNP-470 block copolymerconjugate as described herein.

Assays for the inhibition of catalytic activity of MetAP-2 can beperformed in 96-well microtiter plates. Compounds to be tested aredissolved in dimethyl sulfoxide at 10 mM and diluted ten-fold in assaybuffer (50 mM HEPES, pH 7.4, 100 mM NaCl). Ten microliters of solutionof each compound to be tested for inhibition are introduced into eachcell of the plate. Zero inhibition of enzyme activity is taken to be theresult obtained in cells in which 10 mL of assay buffer was placed. Amixture totaling 90 mL per well and made up of 84 mL of assay buffercontaining 100 mM MnCl₂, 1 mL of L-amino acid oxidase (Sigma Catalog No.A-9378, ˜11 mg/mL), 1 mL of horseradish peroxidase (Sigma Catalog No.P-8451, dissolved in assay buffer at a concentration of 10 mg/mL), 1 mLof the tripeptide Met-Ala-Ser (Bachem) dissolved in assay buffer atconcentration of 50 mM, 1 mL of ortho-dianisidine (Sigma Catalog No.D-1954, freshly made solution in water at a concentration of 10 mg/mL),and MetAP-2 at a final concentration of 1.5 mg/mL are rapidly mixed andadded to each cell containing test or control compound. The absorbenceat 450 nanometers is measured every 20 seconds over a period of twentyminutes using an automatic plate reader (e.g., from Molecular Devices,California, USA). The Vmax in mOD/min, calculated for each well, is usedto represent MetAP-2 activity. The IC₅₀ for each candidate inhibitor isobtained by plotting the remaining activity versus inhibitorconcentrations.

It is noted that some pro-drugs may require pre-treatment (e.g.,enxymatic or non-specific hydrolysis) before assaying for MEtAP-2inhibitory activity.

Fumagillol Derivatives

As one class of MetAP-2 inhibitors, derivatives of fumagillol areformulated for oral administration in compositions and methods describedherein. It should be understood that anywhere in which the instantdescription refers to a MetAP-2 inhibitor, it can also be said to bereferring to a fumagillol derivative having anti-proliferative andanti-angiogenic activity. Fumagillol derivatives useful in thecompositions and methods described herein retain anti-proliferative,including, but not limited to anti-angiogenic, activity—i.e., at least50% of the anti-proliferative activity of TNP-470, as measured in aHUVEC assay (see below). Numerous fumagillol derivatives meeting thiscriterion are known in the art. In one embodiment, suitable fumagillolderivatives for use in the compositions and formulations as disclosedherein are described in U.S. Pat. No. 5,290,807 which is incorporatedherein in its entirety by reference.

In other embodiments, suitable fumagillol derivatives for use in thecompositions and formulations as disclosed herein are representative ofgeneral Formula II as disclosed in U.S. Pat. Nos. 5,166,172; 5,290,807;5,180,738 and 5,164,410, which are hereby incorporated by reference. Infurther embodiments, fumagillol derivatives for use in the compositionsand formulations as disclosed herein are listed in International PatentApplication WO03/027104 which is incorporated herein in its entirety byreference.

In one embodiment, a fumagillol derivative is6-O—(N-chloroacetylcarbamoyl)fumagillol, also known as TNP-470, aderivative of the fumagillol of Formula III of International PatentApplication WO03/027104 shown below.

The structures of fumagillin and 6-O—(N-chloroacetylcarbamoyl)fumagillol(TNP-470) are shown below:

The synthesis of TNP-470 is disclosed in U.S. Pat. Nos. 5,180,738 and5,290,807 which are hereby incorporated herein in their entirety byreference.

Fumagillin has been disclosed to be anti-proliferative and to haveanti-angiogenic activity in U.S. Pat. No. 5,135,919 which isincorporated herein in its entirety by reference. Moreover, variousfumagillol derivatives have been disclosed to be anti-proliferative andto have anti-angiogenic activity in U.S. Pat. Nos. 5,180,738; 5,164,410;5,196,406; 5,166,172; and 5,290,807 which are incorporated herein intheir entirety by reference. Other MetAP-2 inhibitory fumagillolderivatives, such as PPI-2458, are described by Bernier et al., Proc.Natl. Acad. Sci. U.S.A. 101: 10768-10773 (2004), which is incorporatedherein by reference. In particular, one fumagillol derivative(3R,4S,5S,6R)-5-methoxy-4-(2R,3R)-2-methyl-3-(3-methyl-2-butenyl)-oxiranyl)-1-oxaspiro(2,5)oct-6-yl(chloroacetyl)carbamate, also known as 6-O—(N-chloroacetylcarbamoyl) fumagillol orTNP-470 (available from Takeda Chemical Industries, Ltd. of Japan) is aparticularly potent anti-proliferative and anti-angiogenic compound.Bhargava et al. review TNP-470 in Chapter 26 of Angiogenesis in Healthand Disease, G. M. Rubanyi, ed., Marcel/Dekkker: 2000, pp. 387-406. Onecan determine if a fumagillol derivative has anti-proliferative activityusing a proliferation assay as described herein. Similarly, where theproliferation involves angiogenesis, anti-angiogenic activity can bemeasured using an angiogenesis assay as shown in the Examples or asdisclosed herein.

In some embodiments, fumagillol derivatives include, for example, butnot limited to, O-(3,4-dimethoxycinnamoyl)fumagillol;O-(4-methoxycinnamoyl)fumagillol;O-(3,4,5-trimethoxycinnamoyl)fumagillol;O-(4-Chlorocinnamoyl)fumagillol;4-(3,4,5-trimethoxycinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol;O-(4-trifluoromethylcinnamoyl)fumagillol;O-(4-nitrocinnamoyl)fumagillol;O-(3,4-dimethoxy-6-nitrocinnamoyl)fumagillol;O-(4-acetoxycinnamoyl)nimagillol; O-(4-hydroxycinnamoyl)fumagillol;O-(4-acetoxy-3,5-dimethoxycinnamoyl)fumagillol;O-(3,5-dimethoxy-4-hydroxycinnamoyl)fumagillol;4-(4-methoxycinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol;O-(4-dimethylaminocinnamoyl)fumagillol; O-(4-aminocinnamoyl)fumagillol;O-(4-cyanocinnamoyl)fumagillol; O-(3,4,5-trimethoxycinnamyl)fumagillol;O-(4-dimethylaminoethoxycinnamoyl)fumagillol;O-(3-dimethylaminomethyl-4-methoxycinnamoyl)fumagillol;O-(3,4-methylenedioxycinnamoyl)fumagillol;O-(3,4-dimethoxy-6-aminocinnamoyl)fumagillol;O-(4-ethylaminocinnamoyl)fumagillol;O-(4-ethylaminoethoxycinnamoyl)fumagillol;O-(4-dimethylaminocinnamyl)fumagillol; and4-(4-dimethylaminocinnamoyl)oxy-2-(1,2-epoxy-1,5-dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol.

In some embodiments, fumagillol derivatives include derivatives of theformula I, for example, but are not limited to: 1)6-O-(4-methoxyaniline)acetyl fumagillol; 2)6-O-(3,4,5-trimethoxyaniline)acetyl fumagillol; 3)6-O-(2,4-dimethexyaniline)acetyl fumagillol; 4)6-O-(3,4-dimethoxyaniline)acetyl fumagillol; 5)6-O-(3,4-dimethoxy-6-nitroaniline)acetyl 5 fumagillol; 6)6-O-(3,4-dimethexy-6-cyaneaniline)acetyl fumagillol; 7)6-O-(4-allyloxyaniline)acetyl fumagillol; 8)6-O-(4-(2-acetoxyethexy)aniline)acetyl fumagillol; 9)6-O-(3-cyano-4-methoxyaniline)acetyl fumagillol; 10)6-O-(3-(dimethylaminomethyl)-4-methexyaniline)acetyl fumagillol; 11)6-O-(4-(2-methylpropoxyaniline)acetyl fumagillol; 12)6-O-(3-isopropoxy-4-methoxyaniline)acetyl 15 fumagillol; 13)6-O-(4-(N,N-dimethylethoxy)aniline)acetyl fumagillol; 14)6-O-(3,5-diisopropyl-4-methoxyaniline)acetyl fumagillol; 15)6-O-(3,5-dimethyl-4-methoxyaniline)acetyl fumagillol; 16)6-O-(3-isopropyl-4-ethoxy-6-methylaniline)acetyl fumagillol; 17)6-O-(4-propyloxyaniline)acetyl fumagillol; 18) 6-c-(aniline)acetylfumagillol; 19) 6-O-(4-chloroaniline)acetyl fumagillol; 20)6-O-(4-dimethylaminoaniline)acetyl fumagillol; 21)6-O-(4-hydroxyaniline)acetyl fumagillol; 22) 6-O-(4-aminoaniline)acetylfumagillol; 23) 6-O-(3,4-methylenedioxyaniline)acetyl fumagillol; 24)6-O-(4-nitroaniline)acetyl fumagillol; 25)6-O-(2,3,4-trimethoxy-6-aminoaniline)acetyl fumagillol; 26)6-O-(4-acetoxy-3,5-dimethoxyaniline) acetyl fumagillol; 27)6-O-(3,4-dimethoxy-5-hydroxyaniline)acetyl fumagillol; 28)6-O-(4-dimethylaminoethoxyaniline)acetyl fumagillol; 29)6-O-(4-ethylaminoaniline)acetyl fumagillol; 30)6-O-(4-ethylaminoethoxyaniline)acetyl fumagillol; 31)6-O-(3-dimethylaminomethyl-4 methoxyaniline)acetyl fumagillol; 32)6-O-(4-trifluoromethylaniline)acetyl fumagillol; 33) 6-O-(4-acetoxyaniline)acetyl fumagillol; 34) 6-O-(4-cyanoaniline)acetyl fumagillol;35) 6-O-(4-hydroxyethoxyaniline)acetyl fumagillol; 36)6-O-(5-amino-2-methoxypyridine)acetyl fumagillol; 37)6-O-(5-methoxypyrimidine-2-amino)acetyl fumagillol; 38)6-o-(3-methoxy-6-aminopyridazine)acetyl fumagillol; 39)4-((4-methoxyaniline)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol; 40)4-((3,4,5-trimethoxyaniline)acetyl)oxy-2-(1,2 5epoxy-1,5-dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol;41) 6-O-(ethylamino)acetyl fumagillol; 42) 6-O-(isopropyl amino) acetylfumagillol; 43) 6-O-(1-propyl amino)acetyl fumagillol; 44) 6-O-(1-butylamino)acetyl fumagillol; 45) 6-O-(sec-butyl amino)acetyl fumagillol; 46)6-O-(2-methyl-butylamino)acetyl fumagillol; 47) 6-O-(t-butylamino)acetyl fumagillol; 48) 6-O-(pentyl amino)acetyl fumagillol; 49)6-O-(1-methyl-butyl amino)acetyl fumagillol; 50) 6-O-(1-ethyl-propylamino)acetyl-fumagilloli; 51) 6-O-(1-methyl-pentylamino)acetylfumagillol; 52) 6-O-(1,2-dimethyl-butylamino)acetyl fumagillol; 53)6-O-(1,2,2-trimethyl-propylamino)acetyl 20 fumagillol; 54)6-O-(1-isopropyl-2-methylpropylamino)acetyl fumagillol; 55)6-O-(3-methylbutylamino)acetyl fumagillol; 56)6-O-(2-methylallylamino)acetyl fumagillol; 57)6-O-(4-methyl-hepta-2,4-dienylamino)acetyl fumagillol;58)6-O-(1,5-dimethyl-4-hexenylamino)acetyl fumagillol;59)6-O-(1,1-dimethyl-2-propynylamino)acetyl fumagillol; 60)6-O-(prop-2-enylamino)acetyl fumagillol; 61)6-O-(2-bromo-ethylamino)acetyl fumagillol; 62)6-O-(chloroethynylamino)acetyl fumagillol; 63)6-O-(cyclopropylamino)acetyl fumagillol; 64) 6-O-(cyclobutylamino)acetylfumagillol; 65) 6-O-(cyclopentylamino)acetyl fumagillol; 66)6-O-(cyclohexylamino)acetyl fumagillol; 67)6-O-(4-tert-butylcyclohexylamino)acetyl fumagillol; 68)6-O-(2-dimethylamino-1-methylethylamino)acetyl 15 fumagillol; 69)6-O-(2-dimethylamino-propylamino)acetyl fumagillol; 70)6-O-(2-methexy-2-methyl-propylamino)acetyl fumagillol; 71)6-O-(2-oxo-propylamine)acetyl fumagillol; 72)6-O-(1,1-dimethyl-3-oxobutylamino)acetyl fumagillol; 73)6-O-(ethyl-2-aminoacetate)acetyl fumagillol; 74)6-O-(alanine-methylesteramino)acetyl fumagillol; 75)6-O-(methyl-2-amino-3,3-dimethylbutanoate)acetyl fumagillol; 76)6-O-(allylglycine-methylester)acetyl fumagillol; 77)6-O-(2,2-dimethexy-ethylamino)acetyl fumagillol; 78)4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy1,5-dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-15 cyclohexanol; 79)4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5-:dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-1cyclohexanol; and 80) 6-O-(chloro)acetyl fumagillol.

Among the compounds of the Formula I, fumagillol derivatives useful inthe methods and compositions as disclosed herein include, for example,but are not limited to 1) 6-O-(4-methoxyaniline)acetyl fumagillol; 2)6-O-(3,4,5-trimethexyaniline)acetyl fumagillol; 3)6-O-(4-(N,N-dimethylethoxy)aniline)acetyl fumagillol; 4)6-O-(cyclopropylamino)acetyl fumagillol; 5) 6-O-(cyclobutylamino)acetylfumagillol; 6) 4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy-1,5 20dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol; and 7)4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol. Structuralformulas of the above compounds are shown in Tables in InternationalPatent Application No: WO03/027104, which is incorporated herein byreference.

To enhance the activity of anti-proliferative or anti-angiogenictreatments, use of adjunct treatments with MetAP-2 inhibitors, includingfumagillol derivatives, can be performed. Thus in one embodiment, use ofthe compositions as disclosed herein including a MetAP-2 inhibitor blockcopolymer conjugate in conjunction with other agents is encompassed inthe present invention for treatment of diseases, disorders or conditionsassociated with, characterized by, or otherwise requiring MetAP-2activity for their pathologies. These include, but are not limited todiseases involving abnormally stimulated neovascularization, such asinflammatory diseases (rheumatism and psoriasis, among others), diabeticretinopathy and cancer.

Copolymers

One aspect of the invention relates to the use of copolymers as carriersfor the solubilization and formulation of a MetAP-2 inhibitor, forexample TNP-470, as disclosed herein. A copolymer is a polymercomprising subunits of more than one type, i.e a copolymer can comprisesubunits of A and subunits of B etc. In some embodiments, a copolymer isan alternating copolymer, for example, having a repeating structurecomprising the different types of subunits, for example an alternatingcopolymer can have the formula: -A-B-A-B-A-B-A-B-A-B—, or -(-A-B—)_(n)—.

In some embodiments, MetAP-2 inhibitors as disclosed herein areassociated with a block copolymer to form a MetAP-2 inhibitor-blockcopolymer conjugate.

The polymers useful in the compositions and methods described herein areblock copolymers. A block copolymer comprises subunits of more than onetype, but instead of an alternating copolymer, a block copolymercomprises at a minimum, a block of subunits of one subunit type followedby a block of subunits of another subunit type. For example, a blockcopolymer comprising two subunits, for example A and B can have theformula of: -(A-A-)_(n)-(B—B—)_(n)— or -A-A-A-A-A-B—B—B—B—B—. The numberof the different subunits can be different, for example;A-A-A-A-B—B—B—B—B. A diblock copolymer is a block copolymer comprisingtwo different blocks of polymer subunits, whereas a triblock copolymeris a block copolymer comprising three different blocks of polymersubunits, and a tretrablock copolymer is a block copolymer comprisingfour different blocks of copolymer subunits, etc.

As used herein, a block copolymer is a polymer comprising at least afirst block comprising a polymer of hydrophobic monomers, and at least asecond block comprising a polymer comprised of hydrophilic monomers (seeFIG. 14). A “block” copolymer differs from a non-block copolymer in thata block copolymer comprises blocks of polymer of one type (e.g. a blockof hydrophilic monomers) that are joined to a block of polymer ofanother type (e.g. a block of hydrophobic monomers), as opposed to anon-block copolymer in which the different monomers are not joinedtogether in blocks. For example, for a block copolymer of monomers A andB, the block copolymer would have, e.g., the structure AAAAABBBBB. Anon-block copolymer of the same monomer subunits would have, forexample, the structure ABAABABBABAABB or the structure ABABABABABAB, forexample. The blocks of a block copolymer as the term is used herein willhave at least 5 monomers per block (i.e., for a block copolymer of A andB monomers, the A block will be at least 5 A monomers long, and the Bblock will be at least 5 B monomers long). In some embodiments thehomopolymer blocks will be at least 10 monomers long, 15 monomers long,20 monomers long or more. A block copolymer as the term is used hereincan have different block lengths (but each block will be at least 5monomers long)—differences in block lengths can influence the ability ofthe block copolymer to form certain structures, e.g., micelles. In someinstances, the monomers making up the hydrophilic block of a blockcopolymer can comprise different hydrophilic monomer subunits, andsimilarly, in some embodiments, monomers making up the hydrophobic blockof a block copolymer can comprise different hydrophobic monomersubunits.

Diblock copolymers can be made using living polymerization techniques,such as atom transfer free radical polymerization (ATRP), reversibleaddition fragmentation chain transfer (RAFT), ring-opening metathesispolymerization (ROMP), and living cationic or living anionicpolymerizations.

Block copolymers can “microphase separate” to form periodicnanostructures, for example, when one block is hydrophobic and the otherhydrophilic. Microphase separation is a situation similar to that of oiland water. Oil and water don't mix together—they macrophase separate. Ifyou have an “oil-like” first block and a “water-like” second block, theblock copolymers undergo microphase separation. The blocks want to getas far from each other as possible, but they are covalently bonded, sothey're not going to get very far. In “microphase separation” the “oil”and “water” blocks form nanometer-sized structures, including micelles,which comprise essentially spherical arrangement with the hydrophilicblocks arrange to the outside of the sphere, in contact with an aqueoussolution and the hydrophobic blocks forming an inner hydrophobic core.Thermodynamic terms can describe how the different blocks interact. Theinteraction parameter, “chi” gives an indication of how different,chemically, the two blocks are and whether or not they will microphaseseparate. Generally, if the product of chi and the molecular weight islarge (greater than 10.5), the blocks will microphase separate.Conversely, if the product of chi and the molecular weight is too small(less than 10.5), the different blocks are able to mix, rather thanmicrophase separate.

In some embodiments, amphiphilic block copolymers are useful in theformulations and compositions of the present invention, for example aseffective drug carriers that solubilize hydrophobic drugs into anaqueous environment. For example, amphiphilic block copolymersexhibiting self-association properties are disclosed in EP No. 0397 307and EP0583955 and EP0552802, which are incorporated herein by reference.

In some embodiments, useful biodegradable polyesters comprised by thehydrophobic block of copolymers described herein are, for example,biodegradable polyester oligomers or polymers synthesized from monomersselected from, e.g., D,L-lactide, D-lactide, L-lactide, D,L-lactic acid,D-lactic acid, L-lactic acid, glycolide, glycolic acid, ε-caprolactone,ε-hydroxy hexanoic acid, γ-butyrolactone, γ-hydroxy butyric acid,δ-valerolactone, δ-hydroxy valeric acid, hydroxybutyric acids, malicacid, and copolymers thereof. More preferably, the biodegradablepolyester is synthesized from monomers selected from the groupconsisting of D,L-lactide, D-lactide, L-lactide, D,L-lactic acid,D-lactic acid, L-lactic acid, glycolide, glycolic acid, ε-caprolactone,ε-hydroxy hexanoic acid, and copolymers thereof. Most preferably, thebiodegradable polyester is synthesized from monomers selected from thegroup consisting of D, L-lactide, D-lactide, L-lactide, D,L-lactic acid,D-lactic acid, L-lactic acid, glycolide, glycolic acid, and copolymersthereof.

In some embodiments, the block copolymers comprise hydrophobicpolyesters containing polyester bonds, for example but not limited topolylactic acid(PLA), polyglycolic acid(PGA),poly(D,L-lactic-co-glycolic acid)(PLGA), poly(caprolactone),poly(valerolactone), poly(hydroxybutyrate) and poly(hydroxyvalerate).

In some embodiments, the block copolymers comprise hydrophobic polymers,for example of the block copolymer is selected from the group consistingof poly(d,L-lactic acid), poly(L-lysine), poly(aspartic acid),poly(caprolactone) (PCL), poly(propylene oxide). In some embodiments, ahydrophobic polymer moiety is 1-15 kDa. For example, a hydrophobicpolymer moiety is between 0.5-10 kDa, 1-8 kDa, 1-5 kDa, 1-3 kDa, 3-15kDa, 5-15 kDa, 8-15 kDa, 10-15 kDa, 12-15 kDa, 2-12 kDa, 4-10 kDa, 6-8kDa in size. In some embodiments, a hydrophobic polymer is approximately2 kDa, for example 1.5 kDa, 2 kDa, or 2.5 kDa. In particularembodiments, a hydrophobic moiety useful in the composition as disclosedherein is a poly(d,L-lactic acid) (PLA) polymer, for example apoly(d,L-lactic acid) (PLA) polymer of 1 kDa (1000 Da).

In some embodiments, the copolymers comprise hydrophilic polymers, forexample but not limited to polyethene glycol (PEG) polymer (which isalso referred to a poly(ethylene oxide) (PEO) or poly(oxyethelene) inthe art). In some embodiments, the hydrophillic polymer may be capped atone end. In some embodiments, the capping group is alkoxy. For example,a hydrophilic polymer useful in the composition as disclosed by becapped with a methoxy group. In some embodiments, a hydrophilic polymeris between 1-15 kDa. For example, a hydrophilic polymer moiety useful inthe composition as disclosed is between 1-10 kDa, 1-8 kDa, 1-5 kDa, 1-3kDa, 3-15 kDa, 5-15 kDa, 8-15 kDa, 10-15 kDa, 12-15 kDa, 2-12 kDa, 4-10kDa, 6-8 kDa in size. In some embodiments, a hydrophilic polymer isapproximately 2 kDa, for example 1.5 kDa, 2 kDa, or 2.5 kDa. Inparticular embodiments, a hydrophilic polymer is a poly(ethylene glycol)(PEG) polymer, for example a poly(ethylene glycol) (PEG) polymer of 2kDa (2000 Da).

In some embodiments, the copolymers comprise a diblock copolymercomprising a PEG-PLA diblock copolymer, where block copolymer comprisesblocks of the hydrophilic PEG monomers and blocks of the hydrophobicmonomer PLA.

The hydrophilic blocks of a copolymer can be coupled to the hydrophobicblocks by covalent bonds, for example by ester or urethane links and thelike. Condensation polymerization and ring opening polymerizationprocedures may be utilized as may the coupling of a monofunctionalhydrophilic block to either end of a difunctional hydrophobic block inthe presence of coupling agents such as isocyanates. Furthermore,coupling reactions may follow activation of functional groups withactivating agents, such as carbonyl diimidazole, succinic anhydride,N-hydroxy succinimide and p-nitrophenyl chloroformate and the like.

In some embodiments, hydrophilic blocks of a copolymer can comprise PEGor derivatized PEG monomers of an appropriate molecular weight. PEG hasparticularly favorable biocompatibility, nontoxic properties,hydrophilicity, solubilization properties, and rapid clearance from apatient's body.

The hydrophobic blocks of a copolymer should also comprise biodegradablepolyester momomers that are biodegradable and biocompatible. The invitro and in vivo degradation of hydrophobic, biodegradable polyesterblocks of a copolymer are well understood and the degradation productsare readily metabolized and/or eliminated from the patient's body.

MetAP-2 inhibitors, including fumagillol derivatives, such as TNP-470,can be solubilized or dispersed using block copolymers as disclosedherein. One advantage of using the block copolymers as disclosed hereinis that the MetAP-2 inhibitors such as TNP-470 that have limitedsolubility or dispersibility in an aqueous or hydrophilic environmenthave enhanced solubility and/or dispersibility and can be administeredvia oral administration. Another advantage of using the block copolymeris improved biodistribution versus the bengamide class of MetAP-2inhibitors, or the sulphonamide class, or the bestatin class ofinhibitors.

One can prepare block copolymer, MetAP-2 conjugates as micelles asdisclosed in the Examples herein. In another embodiment, one can prepareblock copolymer micelles for sustained release of a MetAP-2 inhibitorusing the method is disclosed in U.S. Pat. No. 6,623,729, which isincorporated in its entirety herein by reference and is furtherillustrated in the following steps:

Step 1: Preparation of Block Copolymer.

A block copolymer containing a hydrophobic part having hydroxyl group atone end and a hydrophilic part at the other end is prepared bycopolymerization of a biodegradable polyester polymer and a polyethyleneglycol(PEG) polymer in the presence of stannous octate as a catalyst:The copolymerization is performed at 160-200.quadrature for 2-6 hoursunder a vacuum condition. The polyester polymer includes polylacticacid(PLA), polyglycolic acid(PGA), poly(D,L-lactic-co-glycolicacid)(PLGA), poly(caprolactone), poly(valerolactone), poly(hydroxybutyrate) or poly(hydroxy valerate), and in some embodiments PLA andmethoxypolyethyleneglycol is used as the polyethyleneglycol polymer.

Step 2: Binding of Linker by an Activation of Functional Groups of BlockCopolymer.

The block copolymer is dissolved in an organic solvent and reacted witha linker at room temperature in the presence of pyridine and nitrogen:The organic solvent includes, but without limitation, methylenechloride,and the linker includes p-nitrophenyl chloroformate,carbonyldiimidazole(CDI), N,N′-disuccinimidyl carbonate(DSC), or amixture of these compounds, preferably p-nitrophenyl chloroformate. Thereaction is carried out for 2 to 6 hours, with a molar ratio of blockcopolymer:linker:pyridine ranging from 1:2:2 to 1:2:6.

Step 3: Preparation of a Conjugate of Drug and Biodegradable Polymer.

The linker-bound block copolymer is conjugated to a drug by covalentlinkage to obtain a micelle monomer of a conjugate of drug and blockcopolymer, where the block copolymer obtained by reacting with hydrazinemay be used: The block copolymer reacted with hydrazine forms a micellemonomer by binding the linker to a ketone group of a MetAP-2 inhibitor,such as a fumagillol derivative, while the block copolymer withouthydrazine reaction forms a micelle monomer by binding the linker to anamine group of a MetAP-2 inhibitor, such as a fumagillol derivative.Preferably, the MetAP-2 inhibitor is TNP-470. In alternativeembodiments, other MetAP-2 inhibitors are equally suitable for copolymerconjugation. In yet another embodiment, other drugs or chemotherapeuticagents are used in addition to a MetAP-2 inhibitor, for exampleanti-cancer agents such as, but without limitation, doxorubicin,adriamycin, cisplatin, taxol and 5-fluorouracil.

Step 4: Preparation of Sustained Release Micelle.

The micelle monomers prepared in Step 3 are dispersed in an aqueoussolution to prepare sustained release micelles. When micelle monomersare dispersed in a certain concentration, micelles are formedspontaneously by thermodynamic equilibrium. Sustained release micellesthus prepared release a drug by way of hydrolysis and enzymatic actionin vivo, and the released drug exerts the same effect as free drug does.

Conjugation of MetAP-2 Inhibitors with Copolymers

In the composition and methods disclosed herein, a MetAP-2 inhibitor,for example TNP-470, is associated with a block copolymer.

As used herein, the term “associated with” means that one entity is inphysical association or contact with another. Thus, a MetAP-2 inhibitor“associated with” a block copolymer can be either covalently ornon-covalently joined to the block copolymer. It is preferred that theassociation be covalent. The association can be mediated by a linkermoiety, particularly where the association is covalent. The term“association” or “interaction” or “associated with” are usedinterchangeably herein and as used in reference to the association orinteraction of a MetAP-2 inhibitor, e.g., TNP-470 with a blockcopolymer, refers to any association between the MetAP-2 inhibitor andthe block copolymer, for example a diblock copolymer comprising ahydrophilic polymer moiety and a hydrophobic polymer moiety, either by adirect linkage or an indirect linkage.

An indirect linkage includes an association between a MetAP-2 inhibitorand the block copolymer, wherein the MetAP-2 inhibitor and blockcopolymer are attached via a linker moiety, e.g., they are not directlylinked. Linker moieties include, but are not limited to, chemical linkermoieties. In some embodiments, a linker between the MetAP-2 inhibitorand the copolymer is formed by reacting the polymer and a linkerselected e.g., from the group consisting of p-nitrophenyl chloroformate,carbonyldiimidazole(CDI), N,N′-disuccinimidyl carbonate(DSC),cis-aconitic anhydride, and a mixture of these compounds.

A direct linkage includes any linkage wherein a linker moiety is notrequired. In one embodiment, a direct linkage includes a chemical or aphysical interaction wherein the two moieties, i.e. the a MetAP-2inhibitor and the block copolymer interact such that they are attractedto each other. Examples of direct interactions include covalentinteractions, non-covalent interactions, hydrophobic/hydrophilic, ionic(e.g., electrostatic, coulombic attraction, ion-dipole,charge-transfer), Van der Waals, or hydrogen bonding, and chemicalbonding, including the formation of a covalent bond. Accordingly, in oneembodiment, a MetAP-2 inhibitor, such as TNP-470 and the block copolymerare not linked via a linker, e.g., they are directly linked. In afurther embodiment, a MetAP-2 inhibitor and the block copolymer areelectrostatically associated with each other.

In one embodiment, the linker is a peptide linker. In anotherembodiment, the peptide linker is enzymatically cleavable, e.g., by aprotease enzyme that cleaves the linkage to release the MetAP-2inhibitor. Most preferably, the peptide linkage is capable of beingcleaved by preselected cellular enzymes, for instance, those found inlysosomes of cancerous cells or proliferating endothelial cells.Alternatively, an acid hydrolysable linker could comprise an ester oramide linkage and be for instance, a cis-aconityl linkage. A pHsensitive linker can also be used. Cleavage of the linker of theconjugate results in release of active MetAP-2 inhibitor. Thus theMetAP-2 inhibitor must be conjugated with the polymer in a way that doesnot alter the activity of the agent. The linker preferably comprises atleast one cleavable peptide bond. Preferably the linker is an enzymecleavable oligopeptide group preferably comprising sufficient amino acidunits to allow specific binding and cleavage by a selected cellularenzyme. Preferably the linker is at least two amino acids long, morepreferably at least three amino acids long. Cleavable linkers aredescribed, e.g., in U.S. Pat. No. 7,332,523, which is incorporatedherein by reference. An example of a preferred peptide linker isGly-Phe-Leu-Gly (see U.S. Pat. No. 7,332,523). Other linker are known tothose of skill in the art.

Pharmaceutical Compositions and Administration:

The MetAP-2 inhibitor in the formulations and compositions as disclosedherein is particularly useful in methods of treating conditions in whichMetAP-2 activity is involved in or required for the pathology of thecondition, including, but not necessarily limited to conditions in whichMetAp-2 is either over-expressed or over-active in a mammal, for examplea human. Such conditions, herein also referred to as a“MetAP-2-dependent disease or disorder” are selected from a groupincluding, but not necessarily limited to cancer, ascites formation,psoriasis, age-related macular degeneration, thyroid hyperplasia,preeclampsia, rheumatoid arthritis and osteoarthritis, Alzheimer'sdisease, obesity, pleural effusion, atherosclerosis, endometriosis,diabetic/other retinopathies, neovascular glaucoma, age-related maculardegeneration, hemangiomas, and corneal neovascularization, HIV, HPV,herpes and other viral infections, anthrax, IPF, COPD, multiplesclerosis, other sclerotic diseases, transplant rejection, lupus, asthmaand other conditions described herein as associated with MetAP-2activity.

In one embodiment, a MetAP-2-dependent disease or disorder is cancer,where the cells are rapidly dividing neoplastic cancer cells, and wherethe neoplastic cells require an efficient blood supply to maintaincontinued growth of the tumor. As used herein, cancer refers to any ofvarious malignant neoplasms characterized by the proliferation ofanaplastic cells that tend to invade surrounding tissue and metastasizeto new body sites and also refers to the pathological conditioncharacterized by such malignant neoplastic growths. The blood vesselsprovide conduits to metastasize and spread elsewhere in the body. Uponarrival at the metastatic site, the cancer cells then work onestablishing a new blood supply network. Administration of a MetAP-2inhibitor in the formulations and compositions as disclosed herein isuseful to inhibit proliferation at the primary disease site, and in thecase of cancer, at secondary tumor sites; embodiments of the inventionserve to prevent and limit the progression of the disease. Any diseasethat requires a continuous proliferation of cells, either primary cells,endothelial cells, adjacent cells or others in order to perpetuate thedisease is a candidate target. For example, candidates for the treatmentof cancer as described herein include, but are not limited to carcinomasand sarcomas found in the anus, bladder, bile duct, bone, brain, breast,cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head andneck, liver, kidney, larynx, lung, mediastinum (chest), mouth, ovaries,pancreas, penis, prostate, skin, small intestine, stomach, spinalmarrow, tailbone, testicles, thyroid and uterus. The types of carcinomasinclude papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor,teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma,rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma,lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, largecell undifferentiated carcinomas, basal cell carcinoma and sinonasalundifferentiated carcinoma. The types of sarcomas include soft tissuesarcoma such as alveolar soft part sarcoma, angiosarcoma,dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor,extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma,hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibroushistiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, andAskin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor),malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, andchondrosarcoma. Abnormal build up and growth of blood vessels in theskin or internal organs in the form of hemangiomas can also be treatedaccording to the methods described herein.

In one embodiment, a MetAP-2-dependent disease or disorder isage-related macular degeneration. It is known that VEGF contributes toabnormal blood vessel growth from the choroid layer of the eye into theretina, similar to what occurs during the wet or neovascular form ofage-related macular degeneration. Macular degeneration, often called AMDor ARMD (age-related macular degeneration), is the leading cause ofvision loss and blindness in Americans aged 65 and older. In order fornew blood vessels to grow (neovascularization) beneath the retina andleak blood and fluid, endothelial cells must proliferate. Thisproliferation is uneven, and results in the leakage described above,which causes permanent damage to light-sensitive retinal cells, whichdie off and create blind spots in central vision or the macula.

In one embodiment, a MetAP-2-dependent disease or disorder is diabeticretinopathy-abnormal blood vessel growth associated with diabetic eyediseases. In diabetic retinopathy and retinopathy of prematurity (ROP)VEGF is released which promotes blood vessel formation—thus, the diseaseor disorder is an angiogenic disease or disorder. Released by the retina(light-sensitive tissue in back of the eye) when normal blood vesselsare damaged by tiny blood clots due to diabetes, VEGF turns on itsreceptor, igniting a chain reaction that culminates in new blood vesselgrowth. However, the backup blood vessels, having proliferated tooquickly and unevenly, are faulty, so they leak, bleed and encourage scartissue that detaches the retina, resulting in severe loss of vision.Such growth is the hallmark of diabetic retinopathy, the leading causeof blindness among young people in developed countries. In oneembodiment, the subject in need of treatment can be a mammal, such as adog or a cat, preferably a human.

In one embodiment, a MetAP-2-dependent disease or disorder is rheumatoidarthritis.⁴⁴ Rheumatoid arthritis (RA) is characterized by synovialtissue swelling, leukocyte ingress and new blood vessel growth fromexisting vessels. The disease is thought to occur as an immunologicalresponse to an as yet unidentified antigen. The expansion of thesynovial lining of joints in rheumatoid arthritis (RA) and thesubsequent invasion by the pannus of underlying cartilage and bonenecessitate an increase in the vascular supply to the synovium, to copewith the increased requirement for oxygen and nutrients. Endothelialcell proliferation is now recognized as a key event in the formation andmaintenance of the pannus in RA (Paleolog, E. M., 2002). Thus, RA is anangiogenesis-related disease or disorder. Even in early RA, some of theearliest histological observations are blood vessels. A mononuclearinfiltrate characterizes the synovial tissue along with a luxuriantvasculature. Endothelial cell proliferation is integral to formation ofthe inflammatory pannus and without it, leukocyte ingress could notoccur (Koch, A. E., 2000). Disruption of the formation of new bloodvessels would not only prevent delivery of nutrients to the inflammatorysite, it could also reduce joint swelling due to the additional activityof VEGF, a potent proangiogenic factor in RA, as a vascular permeabilityfactor.

In one embodiment, a MetAP-2-dependent disease or disorder isAlzheimer's disease. Alzheimer's disease (AD) is the most common causeof dementia worldwide. AD is characterized by an excessive cerebralamyloid deposition leading to degeneration of neurons and eventually todementia. The exact cause of AD is still unknown. It has been shown byepidemiological studies that long-term use of non-steroidalanti-inflammatory drugs, statins, histamine H2-receptor blockers, orcalcium-channel blockers, all of which are cardiovascular drugs with ananti-proliferative effects, seem to prevent Alzheimer's disease and/orinfluence the outcome of AD patients. Therefore, it has been speculatedthat in AD endothelial cell proliferation in the brain vasculature mayplay an important role in AD, that is, AD can be an angiogenesis-relatedor -mediated disease. In Alzheimer's disease, the brain endotheliumsecretes the precursor substrate for the beta-amyloid plaque and aneurotoxic peptide that selectively kills cortical neurons. Moreoveramyloid deposition in the vasculature leads to endothelial cellapoptosis and endothelial cell activation which leads toneovascularization. Vessel formation could be blocked by the VEGFantagonist SU 4312 as well as by statins, indicating thatanti-proliferative or anti-angiogenic strategies can interfere withendothelial cell activation in AD (Schultheiss C., el. al., 2006;Grammas P., et. al., 1999) and can be used for preventing and/ortreating AD.

In one embodiment, a MetAP-2-dependent disease or disorder is obesity.It has been shown that the MetAP-2 inhibitor TNP-470 was able to preventdiet-induced and genetic obesity in mice (Ebba Bråkenhielm et. al.,Circulation Research, 2004; 94:1579). TNP-470 reduced vascularity in theadipose tissue, thereby inhibiting the rate of growth of the adiposetissue and obesity development. Obesity is thus an angiogenesis-relatedor -mediated disease or disorder.

In one embodiment, a MetAP-2-dependent disease or disorder isendometriosis. Excessive endometrial angiogenesis is proposed as animportant mechanism in the pathogenesis of endometriosis (Healy, D L.,et. al., 1998). The endometrium of patients with endometriosis showsenhanced endothelial cell proliferation. Moreover there is an elevatedexpression of the cell adhesion molecule integrin vβ3 in more bloodvessels in the endometrium of women with endometriosis when comparedwith normal women. Strategies that inhibit endothelial cellproliferation can be used to treat endometriosis.

In one embodiment, the method of treating the MetAP-2-dependent diseaseor disorder is applicable to the treatment of cancer; the method asdisclosed herein is applicable to all carcinomas, blood-borne cancersand sarcomas. Preferably, the cancer is selected from the groupconsisting of papilloma/carcinoma, choriocarcinoma, endodermal sinustumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma,leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma,glioma, lymphoma/leukemia, squamous cell carcinoma, small cellcarcinoma, large cell undifferentiated carcinomas, basal cell carcinoma,sinonasal undifferentiated carcinoma, soft tissue sarcoma such asalveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoidtumor, desmoplastic small round cell tumor, extraskeletalchondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma,hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma,liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibroushistiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, andAskin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor),malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, andchondrosarcoma, that are found in the anus, bladder, bile duct, bone,brain, breast, cervix, colon/rectum, endometrium, esophagus, eye,gallbladder, head and neck, liver, kidney, larynx, lung, mediastinum(chest), mouth, ovaries, pancreas, penis, prostate, skin, smallintestine, stomach, spinal marrow, tailbone, testicles, thyroid anduterus.

The MetAP-2 inhibitor in the formulations and compositions as disclosedherein is particularly useful in methods of inhibiting cellproliferation, including angiogenesis at a site of tumorigenesis in amammal.⁴⁵ The MetAP-2 inhibitor in the formulations and compositions asdisclosed herein administered at such sites and in such varied waysprevents or inhibits endothelial cell proliferation and blood vesselformation at the site thereby inhibiting the development and growth ofthe tumor. Tumors which may be prevented or inhibited by preventing orinhibiting proliferation with the conjugate include but are not limitedto melanoma, adenocarcinoma, sarcomas, thymoma, lymphoma, lung tumors,liver tumors, colon tumors, kidney tumors, non-Hodgkins lymphoma,Hodgkins lymphoma, leukemias, uterine tumors, breast tumors, prostatetumors, renal tumors, ovarian tumors, pancreatic tumors, brain tumors,testicular tumors, bone tumors, muscle tumors, tumors of the placenta,gastric tumors, metastases and the like.

In some embodiments, the compositions as disclosed herein comprising aMetAP-2 inhibitor can inhibit proliferation of blood vessel endothelialcells, thus having anti-angiogenesis effect or inhibition ofangiogenesis. Accordingly, the compositions as disclosed hereincomprising MetAP-2 inhibitors can be used in methods for treatingangiogenesis-mediated conditions in which MetAP-2 is involved in thepathology, such as inhibiting growth and metastasis of cancer as well astreating other various diseases where inappropriate angiogenesis occursor proliferation of blood vessel endothelial cells occur, for examplebut not limited to, inflammatory diseases, diabetic retinopathy,rheumatoid arthritis, psoriasis. Accordingly, the compositionscomprising MetAP-2 inhibitors as disclosed herein can be used as acancer metastasis inhibitor or therapeutic agent against cancer,inflammatory diseases, diabetic retinopathy, rheumatoid arthritis,psoriasis and other retinopathies such as retinopathy of prematurity.

Actual dosage levels of active ingredients in the pharmaceuticalcompositions as described herein can be varied so as to obtain an amountof the active compound(s) which is effective to achieve the desiredtherapeutic response for a particular subject or patient. The selecteddosage level will depend upon the activity of the particular MetAP-2inhibitor, the type of administration composition (i.e. tablet versusliquid oral administration versus ocular versus topical versus inhaled,for example), the severity of the condition being treated and thecondition and prior medical history of the patient being treated.

The phrase “therapeutically effective amount” of a compound, e.g., aMetAP-2 inhibitor as described herein means a sufficient amount of thecompound to treat disorders, at a reasonable benefit/risk ratioapplicable to any medical treatment. It will be understood, however,that the total daily usage of the compositions and formulations asdisclosed herein will be decided by the attending physician within thescope of sound medical judgment. A “therapeutically effective amount” asthe term is used herein need not eradicate a disease. Rather, atherapeutically effective amount will at least slow progression of adisease (as non-limiting example, the growth of a tumor or neoplasm)relative to progression without the therapeutic agent, for example thecomposition as disclosed herein comprising a MetAP-2-block copolymerconjugate. Thus, it is preferred, but not required that the therapeuticagent actually eliminate the disease.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the compositions and formulations asdisclosed herein which are employed; and like factors well known in themedical arts. For example, it is well within the skill of the art toeither start doses of the compound at levels lower than required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved, or start doses of thecompound at high levels and to gradually decrease the dosage until thedesired effect is achieved, as appropriate for the care of theindividual patient.

The compositions as disclosed herein can also be administered inprophylatically or therapeutically effective amounts. The formulationsand compositions as disclosed herein can be administered along with apharmaceutically acceptable carrier. A prophylatically ortherapeutically effective amount means that amount necessary, at leastpartly, to attain the desired effect, or to delay the onset of, inhibitthe progression of, or halt altogether, the onset or progression of theparticular disease or disorder being treated. Such amounts will depend,of course, on the particular condition being treated, the severity ofthe condition and individual patient parameters including age, physicalcondition, size, weight and concurrent treatment. These factors are wellknown to those of ordinary skill in the art and can be addressed with nomore than routine experimentation. It is preferred generally that amaximum dose be used, that is, the highest safe dose according to soundmedical judgment. It will be understood by those of ordinary skill inthe art, however, that a lower dose or tolerable dose can beadministered for medical reasons, psychological reasons or for virtuallyany other reasons.

The term “effective amount” as used herein refers to the amount oftherapeutic agent of pharmaceutical composition to alleviate at leastsome of the symptoms of the disease or disorder. The term “effectiveamount” includes within its meaning a sufficient amount ofpharmacological composition to provide the desired effect. The exactamount required will vary depending on factors such as the type of tumorto be treated, the severity of the tumor, the drug resistance level ofthe tumor, the species being treated, the age and general condition ofthe subject, the mode of administration and so forth. Thus, it is notpossible to specify the exact “effective amount”. However, for any givencase, an appropriate “effective amount” can be determined by one ofordinary skill in the art using only routine experimentation.

Efficacy of treatment can be judged by an ordinarily skilledpractitioner. As disclosed in the Examples, efficacy can be assessed inanimal models of cancer and tumor, for example treatment of a rodentwith a cancer, and any treatment or administration of the compositionsor formulations that leads to a decrease of at least one symptom of thecancer, for example a reduction in the size of the tumor or a slowing orcessation of the rate of growth of the tumor indicates effectivetreatment.

Efficacy for any given formulation (e.g., MetAP-2 inhibitor associatedwith block copolymer) can also be judged using an experimental animalmodel of cancer, e.g., wild-type mice or rats, or preferably,transplantation of tumor cells akin to that described in the Examplesherein below. When using an experimental animal model, efficacy oftreatment is evidenced when a reduction in a symptom of the cancer, forexample a reduction in the size of the tumor or a slowing or cessationof the rate of growth of the tumor occurs earlier in treated, versusuntreated animals. By “earlier” is meant that a decrease, for example inthe size of the tumor occurs at least 5% earlier, but preferably more,e.g., one day earlier, two days earlier, 3 days earlier, or more.

In addition, the amount of each component to be administered alsodepends upon the frequency of administration, such as whetheradministration is once a day, twice a day, 3 times a day or 4 times aday, once a week; or several times a week, for example 2 or 3, or 4times a week.

As an example only, TNP-470 administration will be described in greaterdetail as a representative example of the administration procedures forall MetAP-2 inhibitors in general. For6-O—(N-chloroacetylcarbamoyl)fumagillol (TNP-470), the followinginformation may serve as a general guideline for administration.Usually, the formulations and compositions as disclosed herein areadministered from once a day to several times a day, for example 2 timesa day, three times a day, or four times a day. In alternativeembodiments, the formulations and compositions as disclosed herein canbe administered, for example three to five times a week, if it is to beplurally administered in a given week. In some modes of administration,e.g., IV administration, it is desirable to dose less frequently, e.g.,weekly or biweekly; block copolymer conjugate compounds can be useful insuch a regimen.

For example, in one embodiment a suitable dose of the MetAP-2 inhibitorin the formulations and compositions as disclosed herein for a subjectin need of treatment can be used according to conventionally used doseranges of about 1 mg to about 2000 mg TNP-470 equivalent per kilogram ofbody weight. Generally however, conventional doses of fumagillolderivatives are about 0.1 mg/kg to 40 mg/kg body weight, preferablyabout 0.5 mg/kg to 20 mg/kg body weight as disclosed in U.S. Pat. No.5,290,807. In alternative embodiments, where maintenance of tumor growthis the goal (i.e. the goal is to attenuate the growth of the tumor), adose below the threshold used for chemotherapy can be used. For example,a suitable dose could be less than the conventionally usedchemotherapeutic dose, for example, dose ranges of about 1 μg to 1 mg or0.1 μg to 1 mg, or 1 mg to 10 mg TNP-470 per kilogram of body weight canbe used.

In some embodiments, if TNP-470 is administered once a week, it can beadministered in an amount of from about 20 to about 200 mg/m²/week;preferably in an amount of from about 40 to about 180 mg/m²/week; andmost preferably in an amount of from about 135 to about 175 mg/m²/week.In some embodiments, if TNP-470 is administered daily, it may beadministered in an amount of from about 1 to about 10 mg/m²/day; forexample in an amount of from about 1.25 to about 5 mg/m²/day; or in anamount of from about 1 to about 3 mg/m²/day. For continuousadministration, the component is usually administered for at least fiveconsecutive days of the week. In some embodiments, the effective amountof a composition as disclosed herein comprising a fumagillol derivativecan be determined using an anti-angiogenesis assay as disclosed herein,and in some embodiments, the effective amount is less than the amountused as the conventionally effective dose. Similar dosage regimes can beapplied for formulations of other MetAP-2 inhibitor compositions asdisclosed herein.

In an alternative embodiment, higher dosages can be used, provided thereis not unacceptable toxicity. For example, dosages in the range of about50 to about 500 mg/m²/week or more, and sub-ranges within this range arespecifically contemplated. Thus, dosages in the range of about 120 to350 mg/m²/week, 200 to 400 mg/m²/week, etc. are specificallycontemplated.

The preferred route of administration of the compositions andformulations as disclosed herein is oral administration. Solid dosageforms for oral administration include, for example but not limited tocapsules, tablets, pills, powders and granules. In such solid dosageforms, the compositions as disclosed herein may be mixed with at leastone inert, pharmaceutically acceptable excipient or carrier, such assodium citrate or dicalcium phosphate and/or a) fillers or extenderssuch as starches, lactose, sucrose, glucose, mannitol and silicic acid;b) binders such as carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose and acacia; c) humectants such asglycerol; d) disintegrating agents such as agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain silicates and sodiumcarbonate; e) solution retarding agents such as paraffin; f) absorptionaccelerators such as quaternary ammonium compounds; g) wetting agentssuch as cetyl alcohol and glycerol monostearate; h) absorbents such askaolin and bentonite clay and i) lubricants such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate and mixtures thereof. In the case of capsules, tablets andpills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The active components can also be in micro-encapsulated form,if appropriate, with one or more of the above-mentioned excipients. Inthe preparation of pharmaceutical formulations as disclosed herein inthe form of dosage units for oral administration the compound selectedcan be mixed with solid, powdered ingredients, such as lactose,saccharose, sorbitol, mannitol, starch, arnylopectin, cellulosederivatives, gelatin, or another suitable ingredient, as well as withdisintegrating agents and lubricating agents such as magnesium stearate,calcium stearate, sodium stearyl fumarate and polyethylene glycol waxes.The mixture is then processed into granules or pressed into tablets.

In addition, compositions for topical (e.g., oral mucosa, respiratorymucosa) and/or oral administration can form solutions, suspensions,tablets, pills, capsules, sustained-release formulations, oral rinses,or powders, as known in the art are described herein. The compositionsalso can include stabilizers and preservatives. For examples ofcarriers, stabilizers and adjuvants, University of the Sciences inPhiladelphia (2005) Remington: The Science and Practice of Pharmacy withFacts and Comparisons, 21st Ed. The compositions can also be inhaled(pulmonary, nasal), ocular (eyedrop), sub-lingual, suppository, ortopical (e.g., an ointment).

To enhance the activity of anti-proliferative treatments, use of adjuncttreatments is contemplated. In particular, MetAP-2 inhibitors such asTNP-470 have been tested in conjunction with treatment with variousother drugs to enhance efficacy for treatment of diseases induced byabnormally stimulated neovascularization, such as inflammatory diseases(rheumatism and psoriasis among others), diabetic retinopathy, cancerand other diseases and conditions as discussed elsewhere herein.

Soft gelatin capsules can be prepared with capsules containing a mixtureof the active compound or compounds of the invention in vegetable oil,fat, or other suitable vehicle for soft gelatin capsules. Hard gelatincapsules can contain granules of the active compound. Hard gelatincapsules can also contain the targeted delivery composition includingthe targeting moiety and the carrier particle as well as the therapeuticagent in combination with solid powdered ingredients such as lactose,saccharose, sorbitol, mannitol, potato starch, corn starch,arnylopectin, cellulose derivatives or gelatin.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirs. Inaddition to the active components, the liquid dosage forms may containinert diluents commonly used in the art such as, for example, water orother solvents that are compatible with the maintenance of a micelle ofa diblock copolymer as described herein. Liquid preparations for oraladministration can also be prepared in the form of syrups orsuspensions, e.g. solutions or suspensions containing from 0.2% to 20%by weight of the active ingredient and the remainder consisting of sugaror sugar alcohols and a mixture of ethanol, water, glycerol, propyleneglycol and polyethylene glycol provided that such solvent is compatiblewith maintaining the micelle form. If desired, such liquid preparationscan contain coloring agents, flavoring agents, saccharin andcarboxymethyl cellulose or other thickening agents. Liquid preparationsfor oral administration can also be prepared in the form of a dry powderto be reconstituted with a suitable solvent prior to use.

Besides inert diluents, the oral compositions may also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring and perfuming agents

Transdermal patches may also be used to provide controlled delivery ofthe formulations and compositions as disclosed herein to specificregions of the body. Such dosage forms can be made by dissolving ordispensing the component in the proper medium. Absorption enhancers canalso be used to increase the flux of the compound across the skin. Therate can be controlled by either providing a rate-controlling membraneor by dispersing the compound in a polymer matrix or gel. Suchtransdemal patches are useful for treating parts of the body whereabnormally stimulated neovascularization occurs, such as inflammatorydiseases, for example rheumatism and psoriasis among others, diabeticretinopathy and cancer, for example skin cancer or other skin relatedneovascular conditions (such as psoriasis) or malignancies.

A further form of topical administration is to the eye, for example as atreatment for retinopathy, such as diabetic retinopathy or retinopathyof prematurity (ROP) or for the treatment of immune-mediated conditionsof the eye such as autoimmune diseases, allergic or inflammatoryconditions, and corneal transplants. Components of the invention may bedelivered in a pharmaceutically acceptable ophthalmic vehicle, such thatthe component is maintained in contact with the ocular surface for asufficient time period to allow the component to penetrate the cornealand internal regions of the eye, as for example the anterior chamber,posterior chamber, vitreous body, aqueous humor, vitreous humor, cornea,iris/cilary, lens, choroid/retina and sclera. The pharmaceuticallyacceptable ophthalmic vehicle may, for example, be an ointment or anencapsulating material.

In an alternative embodiment, the compositions and formulations asdisclosed herein can be also administered via rectal or vaginaladministration. In such embodiments, the compositions and formulationsas disclosed herein can be in the form of suppositories which can beprepared by mixing the compounds of this invention with suitablenon-irritating excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax which are solid at room temperature butliquid at body temperature and therefore melt in the rectum or vaginalcavity and release the active component.

Dosage units for rectal or vaginal administration can be prepared (i) inthe form of suppositories which contain the active substance mixed witha neutral fat base; (ii) in the form of a gelatin rectal capsule whichcontains the active substance in a mixture with a vegetable oil,paraffin oil or other suitable vehicle for gelatin rectal capsules;(iii) in the form of a ready-made micro enema; or (iv) in the form of adry micro enema formulation to be reconstituted in a suitable solventjust prior to administration.

To further protect the active ingredient, the MetAP-2inhibitor-copolymer conjugates described herein can be used in admixtureor in combination with a gastric acid secretion-inhibitor and/or anantacid.

Gastric acid secretion inhibitors include, for example H₂ blockers (e.g.famotidine, cimetidine, ranitidine hydrochloride, etc.) and proton pumpinhibitors (e.g. lansoprazole, omeprazole, etc.). As an antacid,compounds which elevate the intragastric pH level, such as magnesiumcarbonate, sodium hydrogen carbonate, magnesium hydroxide, magnesiumoxide and magnesium hydroxide can be employed. The oral dosage forms ofthe compositions and formulations as disclosed herein can beadministered after the intragastric pH has been increased to alleviatethe influence of gastric acid by the administration of a gastric acidsecretion inhibitor and/or antacid.

Alternatively, compositions and formulations as disclosed herein can bein a form of enteric-coated preparation for oral administrationcomprising a MetAP-2 inhibitor-block copolymer conjugate. In someembodiments, a MetAP-2 inhibitor containing core for coating with anenteric coating film can be prepared using an oleaginous base or byother known formulation methods without using an oleaginous base. Insome embodiments, the compositions and formulations as disclosed hereinin the form of the drug-containing core for coating with a coating agentmay be, for example, tablets, pills and granules.

The excipient contained in the core is exemplified by saccharides, suchas sucrose, lactose, mannitol and glucose, starch, crystalline celluloseand calcium phosphate. Useful binders include polyvinyl alcohol,hydroxypropyl cellulose, macrogol, Pluronic F-68, gum arabic, gelatinand starch. Useful disintegrants include carboxymethyl cellulose calcium(ECG505), crosslinked carboxymethylcellulose sodium (Ac-Di-Sol),polyvinylpyrrolidone and low-substituted hydroxypropyl cellulose(L-HPC). Useful lubricants and antiflocculants include talc andmagnesium stearate.

The enteric coating agent is an enteric polymer which is substantiallyinsoluble in the acidic pH and is at least partially soluble at weakeracidic pH through the basic pH range. The range of acidic pH is about0.5 to about 4.5, preferably about 1.0 to about 2.0. The range of weakeracidic pH through basic pH is about 5.0 to about 9.0, preferably about6.0 to about 7.5. Specifically, cellulose acetate phthalate,hydroxypropylmethylcellulose phthalate, hydroxypropylmethyl acetatesuccinate (Shin-Etsu Chemicals), methacrylic copolymers (Rhon-Pharma,Eudragit® L-30D-55, L100-55, L100, S100, etc.), etc. can be mentioned asexamples of the enteric coating agent. These materials are effective interms of stability, even if they are directly used as entericcompositions.

In the case of forming micelles, for example, spherical microparticleshaving particle diameters of about 0.01 μm to about 1000 μm aregenerated, or more preferably about 0.01 to about 5 μm (about 10 nm toabout 5,000 nm). This formation can be performed by the methods asdisclosed in the Examples or as disclosed in Japanese Patent ApplicationJP-A-223533/1991. In some embodiments, micelles between 50 and 500 nmare useful in the compositions as disclosed herein, for example about50-100 nm, 100-150 nm, 150-200 nm, 200-250 nm, 250-300 nm, 300-350 nm,350-400 nm, 450-500 nm. In some embodiments, the micelles are about 130nm as disclosed in the examples.

The concentration or content of the MetAP-2 inhibitor in the compositioncan be appropriately selected according to the physicochemicalproperties of the composition. When the composition is in a liquid form,the concentration is about 0.0005 to about 30% (w/v) and preferablyabout 0.005 to about 25% (w/v). When the composition is a solid, thecontent is about 0.01 to about 90% (w/w) and preferably about 0.1 toabout 50% (w/w).

If necessary, additives such as a preservative (e.g. benzyl alcohol,ethyl alcohol, benzalkonium chloride, phenol, chlorobutanol, etc.), anantioxidant (e.g. butylhydroxyanisole, propyl gallate, ascorbylpalmitate, alpha-tocopherol, etc.), and a thickener (e.g. lecithin,hydroxypropylcellulose, aluminum stearate, etc.) can be used in thecompositions and formulations as disclosed herein.

It is noted that diblock copolymer conjugates as described generallyneed no further emulsifiers. Nonetheless, if necessary, one can use anadditional emulsifier with the compositions and formulations asdisclosed herein. Examples of emulsifiers that might be used includepharmaceutically acceptable phospholipids and nonionic surfactants. Theemulsifiers can be used individually or in combinations of two or more.The phospholipid includes naturally occurring phospholipids, e.g. eggyolk lecithin, soya lecithin, and their hydrogenation products, andsynthetic phospholipids, e.g. phosphatidylcholine,phosphatidylethanolamine, etc. Among them, egg yolk lecithin, soyalecithin, and phosphatidylcholine derived from egg yolk or soybean arepreferred. The nonionic surfactant includes macro-molecular surfactantswith molecular weights in the range of about 800 to about 20000, such aspolyethylene-propylene copolymer, polyoxyethylene alkyl ethers,polyoxyethylene alkylarylethers, hydrogenated castor oil-polyoxyethylenederivatives, polyoxyethylene sorbitan derivatives, polyoxyethylenesorbitol derivatives, polyoxyethylene alkyl ether sulfate, and so on.The proportion of the emulsifier is selected so that the concentrationin a final administrable composition will be in the range of about 0.1to about 10%, preferably about 0.5 to about 5%.

In addition to the above-mentioned components, a stabilizer for furtherimproving the stability of the compositions and formulations asdisclosed herein, such as an antioxidant or a chelating agent, anisotonizing agent for adjusting the osmolarity, an auxiliary emulsifierfor improving the emulsifying power, and/or an emulsion stabilizer forimproving the stability of the emulsifying agent can be incorporated.The isotonizing agent that can be used includes, for example, gylycerin,sugar alcohols, monosaccharides, disaccharides, amino acids, dextran,albumin, etc. These isotonizing agents can be used individually or incombination, with two or more. An emulsion stabilizer that can be used,which includes cholesterol, cholesterol esters, tocopherol, albumin,fatty acid amide derivatives, polysaccharides, polysaccharide fatty acidester derivatives, etc.

The compositions and formulations as disclosed herein can furthercomprise a viscogenic substance which can adhere to the digestive tractmucosa due to its viscosity expressed on exposure to water. The examplesof the viscogenic substance include, but are not particularly limited aslong as it is pharmaceutically acceptable, such as polymers (e.g.polymers or copolymers of acrylic acids and their salts) andnatural-occurring viscogenic substances (e.g. mucins, agar, gelatin,pectin, carrageenin, sodium alginate, locust bean gum, xanthan gum,tragacanth gum, arabic gum, chitosan, pullulan, waxy starch, sucralfate,curdlan, cellulose, and their derivatives). Furthermore, for controllingthe release of the active drug or for formulation purposes, theadditives conventionally used for preparing the oral compositions can beadded. Example of the additives include excipients (e.g. lactose, cornstarch, talc, crystalline cellulose, sugar powder, magnesium stearate,mannitol, light anhydrous silicic acid, magnesium carbonate, calciumcarbonate, L-cysteine, etc.), binders (e.g. starch, sucrose, gelatin,arabic gum powder, methylcellulose, carboxymethylcellulose,carboxymethylcellulose sodium, hydroxypropylcellulose,hydroxypropylmethylcellulose, polyvinylpyrrolidone, pullulan, dextrin,etc.), disintegrators (e.g. carboxymethylcellulose calcium,low-substituted hydroxypropylcellulose, croscarmellose sodium, etc.),anionic surfactants (e.g. sodium alkylsulfates etc.), nonionicsurfactants (e.g. polyoxyethylene sorbitan fatty acid esters,polyoxyethylene fatty acid esters, polyoxyethylene-castor oilderivatives, etc.), antacids and mucous membrane protectants (e.g.magnesium hydroxide, magnesium oxide, aluminum hydroxide, aluminumsulfate, magnesium metasilicate aluminate, magnesium silicate aluminate,sucralfate, etc.), cyclodextrin and the corresponding carboxylic acid(e.g. maltosyl-beta-cyclodextrin, maltosyl-beta-cyclodextrin-carboxylicacid, etc.), colorants, corrigents, adsorbents, antiseptics, moisteningagents, antistatic agents, disintegration retardants, and so on. Theproportion of these additives can be appropriately selected from therange that can keep the stability and absorption of the basis.

The compositions and formulations as disclosed herein for oraladministration of the present invention may also include flavoringagents. Such agents include, for example, anise oil, lavender oil, lemonoil, orange essence, rose oil, powder green tea, bergamot oil,(alpha[liter]) borneol, Natural Peal Extract AH-10, Sugar, bitteressence, pine flavor etc.

In the case of forming micelles, for example, spherical microparticleshaving particle diameters of about 0.1 nm to about 1000 nm, thisformation can be achieved by methods known in the art (e.g.JP-A-223533/1991). In some embodiments, micelles between 50 and 500 nmare useful in the compositions as disclosed herein, for example about50-100 nm, 100-150 nm, 150-200 nm, 200-250 nm, 250-300 nm, 300-350 nm,350-400 nm, 450-500 nm. In some embodiments, the micelles are about 130nm as disclosed in the examples.

The compositions and formulations as disclosed herein can exhibit potentpharmacological activity with low toxicity such that they are useful asa medicament for prevention and treatment for, interalia,MetAP-2-associated disease in mammals (e.g. mouse, rat, monkey, bovine,canine, human, etc.), the MetAP-2-associated disease including, forexample, (1) inflammatory diseases such as rheumatoid arthritis, (2)diabetic retinopathy, and (3) benign and malignant tumors (e.g. gastriccancer, cancer of the esophagus, duodenal cancer, cancer of the tongue,pharyngeal cancer, brain tumors, neurilemoma, colorectal cancer,non-small-cell lung cancer, small cell carcinoma of the lung, hepaticcarcinoma, renal cancer, cancer of the breast, biliary tract cancer,cancer of the pancreas, cancer of the prostate, cancer of the uterus,carcinoma of the uterine cervix, ovarian cancer, cancer of the urinarybladder, cancer of the skin, malignant melanoma, cancer of the thyroid,sarcomas of bone, hemangioma, hemangiofibroma, retinal sarcoma, cancerof the penis, solid tumors of childhood, Kaposi's sarcoma in AIDS, etc.,inclusive of recurrencies and metastases to other organs), fibroticdiseases such as idiopathic pulmonary fibrosis, or cystic fibrosis. Itis particularly useful when the dosage form of the compositions andformulations as disclosed herein insures an effective bloodconcentration within the range not causing expression of the sideeffects of the active substance in prolonged time, or not contributingto new side effects due to prolonged blood circulation or depoting inorgan tissue for lack of renal or other clearance mechanism.

Generally, no enteric coating is required for the fumagillol-derivativediblock copolymer formulations described herein to be orally effective.It can be advantageous, however, from the standpoint of stability, thatthe compositions and formulations as disclosed herein are filled intocapsule shells coated with an enteric coating agent as mentioned abovefor use as an enteric composition. As the capsule shell, for example,soft capsules (e.g. the product of R. P. Sealer) and hard gelatincapsules are used.

The liquid or solid compositions and formulations as disclosed hereincan be administered orally. In the case of the liquid form, it can bedirectly administered e.g., by drinking an elixir or suspension of thecomposition, or alternatively, into the digestive tract via a catheteror sonde for oral administration or administered in the usual manner inthe unit dosage form of a hard capsule or a soft capsule. In the case ofthe solid form, it can be administered orally as powders, capsules,tablets, or the like in the usual manner. It can also be redispersed ina suitable dispersion medium and administered in a liquid form. Taking apatient of breast cancer (body weight: 50 kg) as an example, the oraldose of the composition as disclosed herein is about 1 mg to about 3g/day, preferably about 10 mg to about 1 g/day, of a MetAP-2 inhibitor.In some embodiments, the oral dose of the composition as disclosedherein is between the ranges of about 25 mg to about 1 g/day, and insome embodiments less than 25 mg to 1 g/day, for example about 10 mg toabout 0.5 g/day, of a MetAP-2 inhibitor.

The compositions and formulations as disclosed herein enhancebiodistribution, stability and uptake properties of MetAP-2 inhibitorsand increase the pharmacological activity thereof, so that betterassurance of therapeutic efficacy is achieved without the need forparenteral administration. The dosage form of the compositions andformulations as disclosed herein is stable and exhibits remarkableinhibiting activity on proliferative activity in oral administration sothat it can be used as clinically advantageous oral medicine.

Suspensions, in addition to the active components, may containsuspending agents, as for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances, and the like.

Proper fluidity can be maintained, for example, by the use of coatingmaterials such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants.

In one embodiment, the delivery is by intranasal administration of thecomposition, especially for use in therapy of the brain and relatedorgans (e.g., meninges and spinal cord). Along these lines, intraocularadministration is also possible. Suitable formulations can be found inRemington's Pharmaceutical Sciences, 16th and 18th Eds., MackPublishing, Easton, Pa. (1980 and 1990), and Introduction toPharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia(1985), each of which is incorporated herein by reference.

In one embodiment, the present invention encompasses combination therapyin which the formulations and compositions as disclosed herein are usedin combination with a chemotherapeutic agent such as Taxol,cyclophosphamide, cisplatin, gancyclovir and the like. Thechemotherapeutic agent may also be included within a micelle asdescribed herein. Such a therapy is particularly useful in situations inwhich the subject or patient to be treated has a large preexisting tumormass which is well vascularized. The chemotherapeutic agent serves toreduce the tumor mass and the conjugate prevents or inhibitsneovascularization within or surrounding the tumor mass. Thechemotherapeutic agent may also be administered at lower doses thannormally used and at such doses may act as an anti-proliferative agent.The second therapy can be administered to the subject before, during,after or a combination thereof relative to the administration of thecompositions as disclosed herein. Anti-proliferative therapies are wellknown in the art and are encompassed for use in the methods of thepresent invention. Therapies includes, but are not limited to analkylating agent, mitotic inhibitor, antibiotic, or antimetabolite,anti-angliogenic agents etc. Such chemotherapy can compriseadministration of CPT-11, temozolomide, or a platin compound.Radiotherapy can include, for example, x-ray irradiation, w-irradiation,γ-irradiation, or microwaves.

The term “chemotherapeutic agent” or “chemotherapy agent” are usedinterchangeably herein and refers to an agent that can be used in thetreatment of cancers and neoplasms, for example brain cancers andgliomas and that is capable of treating such a disorder. In someembodiments, a chemotherapeutic agent can be in the form of a prodrugwhich can be activated to a cytotoxic form. Chemotherapeutic agents arecommonly known by persons of ordinary skill in the art and areencompassed for use in the present invention. For example,chemotherapeutic drugs for the treatment of tumors and gliomas include,but are not limited to: temozolomide (Temodar), procarbazine (Matulane),and lomustine (CCNU). Chemotherapy given intravenously (by IV, vianeedle inserted into a vein) includes vincristine (Oncovin or VincasarPFS), cisplatin (Platinol), carmustine (BCNU, BiCNU), and carboplatin(Paraplatin), Mexotrexate (Rheumatrex or Trexall), irinotecan (CPT-11);erlotinib; oxalipatin; anthracycline-idarubicin and daunorubicin;doxorubicin; alkylating agents such as melphalan and chlorambucil;cis-platinum, methotrexate, and alkaloids such as vindesine andvinblastine.

In another embodiment, the present invention encompasses combinationtherapy in which the formulations and compositions as disclosed hereinare used in combination with, a cytostatic agent, anti-VEGF and/or p53reactivation agent. A cytostatic agent is any agent capable ofinhibiting or suppressing cellular growth and multiplication. Examplesof cytostatic agents used in the treatment of cancer are paclitaxel,5-fluorouracil, 5-fluorouridine, mitomycin-C, doxorubicin, andzotarolimus. Other cancer therapeutics include inhibitors of matrixmetalloproteinases such as marimastat, growth factor antagonists, signaltransduction inhibitors and protein kinase C inhibitors.

In another embodiment, the methods described herein are administered inconjunction with an anti-VEGF agent. Some examples of anti-VEGF agentsinclude bevacizumab (Avastin™), VEGF Trap, CP-547,632, AG13736, AG28262,SU5416, SU11248, SU6668, ZD-6474, ZD4190, CEP-7055, PKC 412, AEE788,AZD-2171, sorafenib, vatalanib, pegaptanib octasodium, IM862, DC101,angiozyme, Sirna-027, caplostatin, neovastat, ranibizumab, thalidomide,and AGA-1470, a synthetic analog of fumagillin (alternate names:Amebacilin, Fugillin, Fumadil B, Fumadil) (A. G. Scientific, catalog#F1028), an angio-inhibitory compound secreted by Aspergillus fumigates.

As used herein the term “anti-VEGF agent” refers to any compound oragent that produces a direct effect on the signaling pathways thatpromote growth, proliferation and survival of a cell by inhibiting thefunction of the VEGF protein, including inhibiting the function of VEGFreceptor proteins. The term “agent” or “compound” as used herein meansany organic or inorganic molecule, including modified and unmodifiednucleic acids such as antisense nucleic acids, RNAi agents such as siRNAor shRNA, peptides, peptidomimetics, receptors, ligands, and antibodies.Preferred VEGF inhibitors, include for example, AVASTIN® (bevacizumab),an anti-VEGF monoclonal antibody of Genentech, Inc. of South SanFrancisco, Calif., VEGF Trap (Regeneron/Aventis). Additional VEGFinhibitors include CP-547,632(3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride;Pfizer Inc., NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, &SU6668 (formerly Sugen Inc., now Pfizer, New York, N.Y.), ZD-6474(AstraZeneca), ZD4190 which inhibits VEGF-R2 and -R1 (AstraZeneca),CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC 412 (Novartis), AEE788(Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; BayerPharmaceuticals and Onyx Pharmaceuticals), vatalanib (also known asPTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptaniboctasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862(glufanide disodium, Cytran Inc. of Kirkland, Wash., USA),VEGFR2-selective monoclonal antibody DC101 (ImClone Systems, Inc.),angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) andChiron (Emeryville, Calif.), Sirna-027 (an siRNA-based VEGFR1 inhibitor,Sirna Therapeutics, San Francisco, Calif.) Caplostatin, solubleectodomains of the VEGF receptors, Neovastat (

terna Zentaris Inc; Quebec City, CA) and combinations thereof.

In yet another embodiment, the present invention encompasses combinationtherapy in which the formulations and compositions as disclosed hereinare used in combination or conjunction with therapeutics, physiotherapyand/or behavioral psychotherapy used in the treatment of rheumatoidarthritis, obesity, endometriosis, idiopathic pulmonary fibrosis (IPF),lupus, and Alzheimer's disease.

For examples of treatments of rheumatoid arthritis, there aretherapeutic drugs that decrease pain and local inflammation includingaspirin and non-steroidal anti-inflammatory drugs or NSAIDS (such asibuprofen or naproxen) and other immunosuppressive drugs that decreasepain and inflammation while decreasing the growth of abnormal synovialtissue (the tissue that lines the inside of the joint). These drugsinclude methotrexate and low doses of corticosteroids (such asprednisone or cortisone). Other medications used to treat rheumatoidarthritis include: anti-malarial medications (such ashydroxychloroquine), gold, sulfasalazine, penicillamine,cyclophosphamide, cyclosporine, minocycline, interleukin receptorantagonist and anti-IL2 antibodies.

In particular embodiments, the formulations and compositions asdisclosed herein are particularly useful for administration inconjunction therapies used for treatment of diseases associated withvascular permeability, such as vascular complications of diabetes suchas non-proliferative diabetic retinopathy and nephropathy, nephroticsyndrome, pulmonary hypertension, burn edema, tumor edema, brain tumoredema, IL-2 therapy-associated edema, and other edema-associateddiseases, as disclosed in International Application No: WO2003/086178and U.S. Patent Applications US2005/0203013 and US2005/0112063 which areincorporated herein in theri entirety by reference. In a particularembodiment, the formulations and compositions as disclosed herein areparticularly useful for administration in conjunction with IL-2 therapy,where the limiting factor of IL-2 therapy is IL-2 therapy-associatededema as disclosed in International Application No: WO2003/086178 andU.S. Patent Applications US2005/0203013 and US2005/0112063.

Treatment for Alzhemier's disease include, but are not be limited to,nonsteroidal anti-inflammatory drugs (NSAIDs), estrogen, steroids suchas prednisone, vitamin E, menantine, donepezil, rivastigmine, tacrine,and galantamine. Holistic medicine include example such as gingko nutsextracts.

Treatment of endometrosis include, but should not be construed aslimited to, a combination oral contraceptives (estrogen plus aprogestin), progestins (such as medroxyprogesterone, danazol (asynthetic hormone related to testosterone, gonadotropin-releasinghormone agonists (GnRH agonists such as buserelin, goserelin, leuprolideand nafarelin), and nonsteroidal anti-inflammatory drugs (NSAIDs) forpain control.

Examples of treatment options for obesity include dieting andnutritional counseling, exercise regime, gastric-bypass surgery, anddrugs such as a combination of fenfluramine and phentermine (oftencalled fen-phen), orlistat, sibutramine, phentermine, benzphetamine,diethylpropion, mazindol, and phendimetrazine.

In alternative embodiments, the formulations and compositions cancomprise a plurality of micelles, wherein some micelles comprise MetAP-2inhibitor, and other micelles comprise other therapeutic agents, forexample chemotherapeutic agents and antineoplastic agents.

The amount of the pharmaceutical composition as disclosed herein whenadministered to a subject will, of course, be dependent on the subjectbeing treated, the severity of the affliction, the manner ofadministration, the judgment of the prescribing physician, etc.

The formulations and pharmaceutical composition can, if desired, bepresented in a suitable container (e.g., a pack or dispenser device),such as an FDA approved kit, which can contain one or more unit dosageforms containing the carrier portion containing the targeting and immuneresponse triggering portions.

Proliferative Assays

In general, one of skill in the art will know whether a given agent, forexample a fumagillol derivative, is an anti-proliferative oranti-angiogenic agent. For the avoidance of doubt, one can use any of anumber of in vitro or in vivo assays to evaluate the influence of agiven agent on proliferation or angiogenesis. Whether or not acomposition or formulation comprising a MetAP-2 inhibitor blockcopolymer conjugate in accordance with the present invention, can treator prevent diseases associated with an angiogenesis-mediated conditioncan be determined by its effect in the mouse model as shown in theExamples below. However, at a minimum, a MetAP-2 inhibitor blockcopolymer as described herein will have anti-proliferative activity(i.e., at least 50% of the anti-proliferative activity of TNP-470) in aHUVEC assay as described in the Examples herein.

Another useful assay for determining if the compositions andformulations has disclosed herein have anti-proliferative oranti-angiogenesis activity is the CAM assay, which is frequently used toevaluate the effects of endothelial cell proliferation regulatingfactors because it is relatively easy and provides relatively rapidresults. A proliferation-regulating factor useful in the methods andcompositions described herein will modify the number of microvessels inthe modified CAM assay described by Iruela-Arispe et al., 1999,Circulation 100: 1423-1431. The method is based on the vertical growthof new capillary vessels into a collagen gel pellet placed on the CAM.In the assay as described by Iruela-Arispe et al., the collagen gel issupplemented with a proliferative factor such as FGF-2 (50 ng/gel) orVEGF (250 ng/gel) in the presence or absence of test agents. The extentof the proliferative response is measured using FITC-dextran (50 μg/mL)(Sigma) injected into the circulation of the CAM. The degree offluorescence intensity parallels variations in capillary density; thelinearity of this correlation can be observed with a range ofcapillaries between 5 and 540. Morphometric analyses are performed, forexample, by acquisition of images with a CCD camera. Images are thenanalyzed by imported into an analysis package, e.g., NHImage 1.59, andmeasurements of fluorescence intensity are obtained as positive pixels.Each data point is compared with its own positive and negative controlspresent in the same CAM and interpreted as a percentage of inhibition,considering the positive control to be 100% (VEGF or FGF-2 alone) andthe negative control (vehicle alone) 0%. Statistical evaluation of thedata is performed to check whether groups differ significantly fromrandom, e.g., by analysis of contingency with Yates' correction.

Additional proliferative assays, including additional assaysspecifically designed to monitor angiogenic proliferation are known inthe art and can be used to evaluate MetAP-2 inhibitors for use in themethods and compositions described herein. These include, for example,the corneal micropocket assay, hamster cheek pouch assay, the Matrigelassay and modifications thereof, and co-culture assays. Donovan et al.describe a comparison of three different in vitro assays developed toevaluate angiogenesis regulators in a human background (Donovan et al.,2001, Angiogenesis 4: 113-121, incorporated herein by reference).Briefly, the assays examined include: 1) a basic Matrigel assay in whichlow passage human endothelial cells (Human umbilical vein endothelialcells, HUVEC) are plated in wells coated with Matrigel (BectonDickinson, Cedex, France) with or without angiogenesis regulator(s); 2)a similar Matrigel assay using “growth factor reduced” or GFR Matrigel;and 3) a co-culture assay in which primary human fibroblasts and HUVECare co-cultured with or without additional angiogenesis regulator(s),the fibroblasts produce extracellular matrix and other factors thatsupport HUVEC differentiation and tubule formation. In the Donovan etal. paper the co-culture assay provided microvessel networks that mostclosely resembled microvessel networks in vivo. However, the basicMatrigel assay and the GFR Matrigel assay can also be used by one ofskill in the art to evaluate whether a given fumagillol derivative is anangiogenesis-inhibiting agent as necessary for the methods andcompositions described herein. Finally, an in vitro angiogenesis assaykit is marketed by Chemicon (Millipore). The Fibrin Gel In VitroAngiogenesis Assay Kit is Chemicon Catalog No. ECM630.

Other proliferative assays are disclosed in International ApplicationNo: WO2003/086178 and U.S. Patent Applications US2005/0203013 andUS2005/0112063, and involve assaying endothelial cells on a permeablesubstrate (e.g., a collagen coated inserts of “Transwells”), contactingthe assay with a test compound (e.g., a fumagillol derivative blockcopolymer conjugate), treating the assay with a marker (e.g., FITClabel) and a permeability-inducing agent (e.g., vascular endothelialgrowth factor (VEGF) and platelet-activating factor (PAP) among others),and measuring the rate of diffusion of the marker compare to control.Compounds that are found to affect vascular permeability can be furthertested for anti-proliferative activity using existing methods. Thebioeffectiveness of MetAP-2 inhibitor block copolymer conjugate as ananti-proliferative or angiogenic agent in a patient being treated withsuch a block copolymer conjugate can be assessed by methods commonlyknown by person skilled in the art, for example, as disclosed inInternational Application No: WO2003/086178 and U.S. Patent ApplicationsUS2005/0203013 and US2005/0112063. One approach involves administeringto the patient an intradermal injection of histamine before treating thesubject with the a MetAP-2 block copolymer conjugate and measuring ahistamine-induced local edema. Then, treating the subject with the aMetAP-2 inhibitor block copolymer conjugate, and again administering tothe subject an intradermal injection of histamine subsequent to treatingthe subject with the MetAP-2 inhibitor block copolymer conjugate andmeasuring the histamine-induced local edema. A decrease in themeasurement of the histamine-induced local edema compared to that seenbefore the treatment with the MetAP-2 inhibitor block copolymerconjugate indicates that the compound is bioeffective.

Composition or formulation as disclosed herein capable of preventing ortreating non-proliferative diabetic retinopathy can be tested by invitro studies of endothelial cell proliferation and in other models ofdiabetic retinopathy, such as Streptozotocin. In addition, color Dopplerimaging can be used to evaluate the action of a drug in ocular pathology(Valli et al., Ophthalmologica 209(13): 115-121 (1995)). Color Dopplerimaging is a recent advance in ulkasonography, allowing simultaneoustwo-dimension imaging of structures and the evaluation of blood flow.Accordingly, retinopathy can be analyzed using such technology. Themanner in which the compositions and formulations as disclosed hereinare administered is dependent, in part, upon whether the treatment of adisease associated with vascular hyperpermeability, includingnon-proliferative retinopathy is prophylactic or therapeutic. Forexample, the manner in which compositions and formulations as disclosedherein are administered for treatment of retinopathy is dependent, inpart, upon the cause of the retinopathy. Specifically, given thatdiabetes is the leading cause of retinopathy, the compositions andformulations as disclosed herein can be administered preventatively assoon as the pre-diabetic retinopathy state is detected.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±1%. The present invention is further explained in detail by thefollowing examples, but the scope of the invention should not limitthereto.

EXAMPLES

The examples presented herein relate to methods, compositions andformulations of MetAP-2 inhibitors for oral or alternativeadministration. Throughout this application, various publications arereferenced. The disclosures of all of the publications and thosereferences cited within those publications in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.The following examples are not intended to limit the scope of the claimsto the invention, but are rather intended to be exemplary of certainembodiments. Any variations in the exemplified methods which occur tothe skilled artisan are intended to fall within the scope of the presentinvention.

Methods

Abbreviations:

mPEG-PLA=methoxy-poly(ethylene glycol)-poly(lactic acid),Suc=succinated; EDC=ethyl(diethylaminopropyl) carbodiimide;NHS=N-hydroxysuccinimide; en=ethylenamine; DDW=Double distilled water;DMF=Dimethylformamide; DMSO=Dimethyl sulfoxide.

Animals

All animal procedures were performed in compliance with BostonChildren's Hospital guidelines, and protocols were approved by theInstitutional Animal Care and Use Committee.

Mice (C57/B16J, 8 weeks old) were purchased from Jackson Laboratories(Bar Harbor, Me., USA). For rat studies, non pigmented Lewis rats (8-10wk old, Charles River Laboratories) were used. All protocols wereapproved by the Institutional Animal Care and Use Committee atChildren's Hospital Boston and were conducted in accordance with theAssociation for Research in Vision and Ophthalmology's Statement for theUse of Animals in Ophthalmic and Vision Research.

Log D Measurement for TNP-470

Aqueous solubility is one of the important chemical properties affectingoral absorption of a drug. In order to predict the intestinal absorptionof TNP-470, we measured its Log-D value which is a parameter ofhydrophobicity determined by the ratio of drug concentration in octanolto that in water at 25° C. (Analiza, Cleveland Ohio). High Log-D values(>2) indicate low water solubility and hence a poor oral availability ofa drug. For this study Log-D values of TNP-470 (30 mM) were measured atplasma and stomach pHs: pH=7.4 and pH=2 respectively.

Preparation of PEG-PLA-MetAP-2 Polymersomes(Polymeric-Micelles).

A known MetAP-2 inhibitor, TNP-470 (Takeda) was chosen for its activity,hydrophobicity and poor pharmacokinetic profile (see Placidi, CancerRes. 55, 3036-3042 (1995)) and was bound to a diblock co-polymer using atwo-step conjugation (FIG. 6A). In the first step succinatedmonomethoxy-poly(ethylene glycol)-poly(lactic acid) (mPEG2000-PLA1000)with free carboxic acid end-groups (Advanced Polymers Materials Inc.),was reacted with ethylenediamine (Sigma-Aldrich). SuccinatedmPEG-b-PLA-OOCCH2CH2COOH (500 mg) was dissolved in DMSO and reacted withethyl(diethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide(NHS) in a molar ratio of 1:10:20 (Polymer:EDC:NHS respectively). In analternative approach, the same amount of succinatedmPEG-b-PLA-OOCCH2CH2COOH is dissolved in 10 ml MES buffer at pH 6 andthe catalyst is N-hydroxysuccinimide (sulfo-NHS)). A five fold molarexcess of ethylendiamine was added and reacted for 4 h at 25° C. Thepolymer solution was then dialyzed (MWCO 1000, Spectra/Por BiotechRegenerated Cellulose VWR) against DMSO leading to a 65% reactionefficiency. In the second step, the amine containing polymer was mixedwith TNP-470 (350 gr), dissolved in DMSO and the solution was stirredfor 4 h at 25° C. The polymeric micelles were formed by dialyzing theDMSO solution of the conjugate against double distilled water using aregenerated cellulose dialysis bag (MWCO 1000, Spectra/Por BiotechRegenerated Cellulose, VWR) to obtain micelles with high incorporationefficiency (>90%) and 0.8-1% free drug (w/w). The micelles were thenlyophilized and stored at −20° C. in a dry environment until use.

Rhodamine labeled polymeric-micelles were formed using a similarprotocol. The mPEG-PLA was conjugated via the N-terminal amine groupwith Lissamine rhodamine B sulfonyl chloride (Molecular Probes, Eugene,Oreg.) in DMSO. For green fluorescent polymer micelles, a commonly usedhydrophobic marker 6-coumarin (Sigma-Aldrich, MO, USA) at 0.1% w/w wasadded to the polymeric solution before the final dialysis step.

For fluorescent labeled Lodamin, a commonly used hydrophobic marker6-coumarin (Sigma-Aldrich) at 0.1% wt/wt was added to the polymericsolution before the final dialysis step.

Characterization of Conjugation by NMR Analysis

Nuclear Magnetic Resonance (NMR) spectrometer analysis (NERCE/BEID,Harvard Medical School) was conducted for each reaction step and Massspectrometry (Proteomic core, Harvard Medical School) was performed onthe conjugate. In order to evaluate the efficiency of ethylendiaminebinding to mPEG-PLA (first reaction step, FIG. 6A) and TNP-470 bindingto the polymer via the amine (second step, FIG. 6A), we used thecolorimetric amine detection reagent: 2,4,6-Trinitrobenozene SulfonicAcid (TNBSA) (Pierce, Rockford Ill.) was used. TNBSA reacts with primaryamines to produce a yellow product whose intensity was measured at 450nm. To calculate amine concentration in the polymer a linear calibrationcurve of amino acid was used. To measure TNP-470 loading intopolymeric-micelles, we incubated 10 mg/ml Lodamin in 500 μl NaOH (0.1 N)to accelerate PLA degradation. After an overnight incubation withshaking (100 r.p.m) at 37° C., we added Acetonitrile to the samples (1:1NaOH:Acetonitrile) and analyzed for TNP-470 concentration. TNP-470concentration was measured using a high-performance liquidchromatography system (HPLC, System Gold® Microbore, Beckman Coulter). A20-μl portion of each sample was injected into a Nova-pak C18 column(3.9-×150-mm i.d.; Waters), and analyzed using a calibration curve ofTNP-470. TNP-470 binding to amine was also measured using TNBSA reagentand was confirmed by subtracting the non-bound drug from the total drugadded to the reaction.

Amine Determination

The first step of ethylenediamine binding to the polymer, and the secondstep of TNP470 conjugation reaction through the free amine, were furtherverified using (2,4,6-Trinitrobenozene Sulfonic Acid (TNBSA) reagent(Pierce, Rockford Ill.). TNBSA is a sensitive assay reagent which cancouple to free amino groups and form a highly chromogenic derivate. Thereaction was carried out after the addition of 0.01% (w/v) TNBSA in 0.1MSodium bicarbonate buffer. A series of PEG-PLA polymer concentrationsbefore and after TNP-470 conjugation were allowed to react for 2 hr in37° C. with TNBSA and the yellow color was measured using a platereader.

The correlation between the molar amine concentration and colorabsorption at 450 nm was determined according to a standard curve donefor aspartic acid.

Cell Culture.

Murine Lewis lung carcinoma (LLC) and B16/F10 melanoma cells wereobtained from American Type Culture Collection (ATCC, Manassas, Va.,USA). Human Umbilical Vascular Endothelial cells (HUVEC) were purchasedfrom Cambrex (USA). The cells were grown and maintained in medium asrecommended by the manufacturers. Dulbecco's Modified Eagle's Mediumwith 10% fetal bovine serum was used for tumor cells and EMB-2 (CambrexBio Science Walkersville, Inc.) containing 2% fetal bovine serum andEGM-2 supplements was used for HUVEC.

RPE cells were cultivated as has been previously described⁵⁹. Asdetermined by flow cytometry, the primary RPE cultures were found to bemore than 98% cytokeratin positive (Clone PCK-26, Sigma). HMVECs, werepurchased from Cambrex Bio Science Inc. (Rockland, Me.) and weremaintained on collagen I coated plates with endothelial cell growthmedium containing 2% FBS (EGM-2; Cambrex, Inc.) at 37° C. withhumidified 95% air/5% CO₂.

HUVEC Proliferation Assay

HUVEC were exposed to different concentrations of Lodamin equivalent to50-1,000 nM free TNP-470 (0.12-2.4 mg/ml micelles) and incubated in alow serum medium for 48 h at 37° C. To rule out a possible cytotoxiceffect of the carrier, empty micelles were added to HUVECs as a control(4.8 mg/ml). A WST-1 proliferation assay (Roche Diagnostics) was used.Cell viability was calculated as the percentage of formazan absorbanceat 450 nm of treated versus untreated cells. Data were derived fromquadruplicate samples in two separate experiments. The effect of Lodamin(60 nM TNP-470 equivalent q.o.d. on HUVEC growth rate was evaluated bydaily counting of HUVEC cells up to 5 d and compared to the number ofuntreated cells or cells treated with Vehicle (same concentration asLodamin).

Uptake of Polymeric Micelles by HUVEC and their Localization in Cells.

To evaluate the uptake of polymeric micelles by HUVEC, 6-coumarinlabeled PEG-PLA micelles or rhodamine conjugated to mPEG-PLA were used.HUVEC were seeded in a 24-well plate (2×10⁴ per well) in EGM-2 medium(Cambrex) and were allowed to attach overnight. Fluorescent-labeledmicelles (10 mg/ml) were suspended over a bath sonicator for 5 min, and20 μl of the suspension was added to the cultured cells. After thedesignated time points (20 min, 2, 4, 7 and 24 h), the cells were washedthree-times with PBS and analyzed by flow cytometry, or alternativelyfixed with 4% parformaldehyde. For confocal microscopy, cells weremounted using DAPI containing Vectashild (Vector, Laboratories,Burlingame, Calif.). Optical sections were scanned using Leica TCS SP2AOBS a ×40 objective equipped with 488-nm argon, 543-nm HeNe, and 405-nmdiode lasers. To study Lodamin internalization into endothelial cells,confocal microscopy was used to co-localize 6-coumarin labeledpolymeric-micelles with endo-lysozome. Live HUVEC cells were imaged indifferent time points after addition of labeled micelles to cell media(15 μg/ml) up to 1 h. At this point, LysoTracker Red® (Molecular Probes,Eugene, Oreg.) was added to the medium for the detection of acidicintracellular vesicles: Endosomes and Lysosomes. After 20 min ofincubation, cells were imaged by confocal using optical sections with488-nm argon, 543-nm HeNe, and 405-nm diode lasers.

To further verify that polymeric micelle internalization occurs throughendocytosis, cell uptake was measured in cold conditions (4° C.) incomparison to cell uptake at 37° C. HUVEC were plated at a concentrationof 15,000 cells/ml in two 24-well plates for 24 h. Fluorescent labeledpolymeric micelles (15 μg/ml) were added and incubated at different timepoints: 20, 30, 40 and 60 min (n=3) in cold room (4° C.) and in 37° C.After the designated time points the cells were washed 3 times with PBSand lysed with 100 μl lysis buffer (BD Biosciences). Cell extracts weremeasured for fluorescent signal in a Wallac 1420 VICTOR plate-reader(Perkin-Elmer Life Sciences) with excitation/emission at 488 nm/530 nm.

Morphology of MetAP-2 Inhibitor Polymeric Micelles by TransmissionElectron Microscopy (TEM).

In order to study the morphology of MetAP-2 inhibitor polymersomes,Transmission Electron Microscopy (TEM) images were taken at the day ofpreparation and one week post preparation. Polymer micelles dispersed indouble distilled water were imaged with cryo-TEM (JEOL 2100 TEM, HarvardUniversity—CNS).

In-Vitro TNP-470 Release from MetAP-2 Inhibitor-Polymersomes.

To study the kinetic release of TNP-470, Lodamin (20 mg) was incubatedwith either 1 ml PBS pH=7.4 or simulated gastric fluid (HCL:ddw pH=1.2).Every few days, supernatant was taken and analyzed for TNP-470concentration and a cumulative release graph of TNP-470 was determined.TNP-470 concentration was measured using HPLC. TNP-470 was detected as apeak at 6 min with 50% acetonitrile in water at the mobile phase. Theflow rate was 1 ml/min, and the detection was monitored at 210 nmwavelength.

Size of MetAP-2 Inhibitor Polymeric Micelles

The average size and size distribution of polymeric micelles weredetermined by Dynamic Light Scatterer (DLS, DynaPro, Wyatt Technology).The measurements were done at 25° C. using Dynamic V6 software. Lodamin(TNP-470 polymersomes) at 1.5 or 2 mg/ml dispersed in double-distilledwater were measured by 20 successive readings with DLS. Micelles' sizewas also measured after 24 hr to evaluate their structural stability.

Evaluation of Antitumor Activity of MetAP-2 Inhibitor PolymersomesIn-Vivo

Animal procedures were performed in the animal facility at Children'sHospital. Eight-week-old male C57BL/6 mice (Jackson Laboratories, BarHarbor, Mass.) were injected subcutaneously with 1×10⁶Lewis LungCarcinoma cells (LLC) at the back. When tumors reached a volume of 100mm³ the mice were divided into 5 groups, and treated every day for 12days with TNP-470 polymeric micelles, administrated orally using agavage needle. Different doses of polymersomes were given daily: 15mg/kg (5 mice), 30 mg/kg (3 mice) and 60 mg/kg (1 mice) TNP-470equivalent, respectively (5, 10, 20 mg polymeric drug per mouse,respectively). The control groups were five mice that were given thesame dose of polymersomes without the drug, and five mice that weregiven drinking water.

Every two days the weight of the mice was monitored and tumorsdimensions were measured using a caliper. Tumor volume was calculatedaccording to an ellipsoid formula.

Absorption Kinetics of Polymeric Micelles in the Gastrointestinal Tract

A non-invasive IVIS 200 in-vivo imaging system (Xenogen system 3.0) wasused in order to track the polymeric micelles labeled with fluorescentmarker in a manner similar to the cell uptake study. Fluorescentpolymersomes were given orally to three nude mice, the mice wereanesthetized using an Isofluran chamber, and images were taken afterdifferent time periods. All the images were taken under identicalconditions.

Corneal Micropocket Assay

In order to evaluate the antiangiogenic properties of Lodamin, thecorneal micropocket angiogenesis assay was performed as previouslydetailed²⁹. Pellets containing 80 ng carrier-free recombinant human bFGFor 160 ng VEGF (R&D Systems, Minneapolis, Minn.) were implanted intomicropockets created in the cornea of anesthetized mice. Mice weretreated daily with 15 mg/kg TNP-470 equivalent of Lodamin for 6 d, andthen the vascular growth area was measured using a slit lamp. The areaof neovascularization was calculated as vessel area by the product ofvessel length measured from the limbus and clock hours around thecornea, using the following equation: Vessel area (mm2)=[π×clockhours×vessel length (mm)×0.2 mm].

Body Distribution, Intestinal Absorption and Toxicity of Lodamin

For all biodistribution studies we used a fluorescent marker fortracking Lodamin. Mice were administered 6-coumarin labeled mPEG-PLA byoral gavage for 3 d (100 μl of 1.5 mg/ml). On the third day oftreatment, after 8 h of fasting, animals were sacrificed and spleen,kidney, brain, lungs, liver, intestine, stomach, and bladder werecollected. The fluorescent 6-coumarin was extracted from the tissues byincubation with formamide for 48 h at 25° C. Samples were centrifugedand signal intensity of fluorescence of supernatants was detected with aWallac 1420 VICTOR plate-reader (Perkin-Elmer Life Sciences) withexcitation/emission at 488 nm/530 nm. The results were normalized toprotein levels in the corresponding tissues. Tissue autofluorescence wascorrected by subtracting the fluorescent signal of non-treated mouseorgans from the respective readings in treated mice. Similarly, levelsof fluorescent signal in mouse sera were measured in different timepoints (1, 2, 4, 8, 24, 48 and 72 h) using excitation/emission readingsat 488 nm/530 nm.

In order to analyze cell uptake in the different tissues in tumorbearing mice, PEG-PLA-rhodamine micelles (100 μl of 1.5 mg/ml) or waterwere orally administered to C57Bl/6J mice bearing LLC tumors (200 mm³)for 3 d. Organs were removed, incubated for 50 min in collagenase(Liberase Blendzyme 3; Roche Diagnostics Corp., IN) in 37° C. to obtaina single cell suspension. These suspensions were analyzed by flowcytometry in order to quantify the uptake of micelles into differenttissue cells when compared to those in the untreated mouse.

In order to evaluate intestinal absorption, mPEG-PLA-rhodamine micelleswere orally administered to C57Bl/6J mice after 8 h of food fasting.After 2 h mice were sacrificed and 2.5 cm segments of the smallintestine were removed, washed, and analyzed by histology and confocalmicroscopy. The rhodamine-labeled polymeric micelles were detected byconfocal microscopy (Leica TCS SP2 AOBS) with a 488-nm argon laser line.Actin filaments were stained with phalloidin-FITC (Sigma) and nucleiwere stained by DAPI (Sigma). To further study the uptake of Lodamin inthe intestine, high resolution images were imaged with cryo-TEM.Intestines from treated (as above) and untreated mice were excised andimmersed immediately in a freshly prepared 4% paraformaldehyde in PBS pH7.4 for 2 h at 25° C. The samples were washed in PBS, transferred to a30% sucrose solution overnight at 4° C. and embedded in OCT and kept at−80° C. until processing. Fifteen sections with 10 μm each were preparedand processed for Confocal microscopy or TEM. Four TEM samples werefixed for 30 min in freshly prepared 2% paraformaldehyde, 2.5%glutaraldehyde, 0.025% CaCl₂ in 0.1 M sodium cacodylate buffer, pH 7.4and subsequently postfixed for 30 min in 1% osmium tetroxide in 0.1 Msym collidine buffer, pH 7.4 at 25° C., stained en bloc in 2% uranylacetate, dehydrated, and embedded under inverted plastic capsules.Samples were snapped free of the glass coverslips by a cycle of rapidfreezing and thawing. Thin sections were cut en face with diamond knivesusing a LEICA UCT Ultramicrotome. Specimens were examined using a JEOL2100 TEM.

To exclude tissue toxicity, histological analysis (H&E) of liver,intestine, lung and kidneys was conducted (Beth-Israel pathologydepartment, BIDMC). To further exclude liver toxicity we analyzed serumlevels of liver enzymes: ALT, AST (Done at Shriners Burns Hospital,Boston, Mass.). These studies were performed on 20 d-long Lodamintreated mice (15 mg/kg TNP-470 equivalent q.d.) and compared tountreated mice (n=3-4).

Oral Administration of Lodamin In-Vivo and Primary Tumor Experiments

Animal procedures were performed in the animal facility at Children'sHospital Boston using 8 week old C57Bl/6J male mice (JacksonLaboratories, Bar Harbor, Me.).

For tumor experiments: LLC cells (1×10⁶) or B16/F10 melanoma cells(1×10⁶) were implanted subcutaneously in 8 week old C57Bl/6J male mice(Jackson Laboratories, Bar Harbor, Me.). Oral availability of freeTNP-470 was compared to that of Lodamin. A dose of 30 mg/kg q.o.d. offree TNP-470 and an equivalent dose of Lodamin (Lodamin, 6 mg in 100μl/d) were administered to LLC tumor bearing mice (˜100 mm³) and tumorgrowth was followed for 18 d. Free drug was given as a suspension indouble distilled water and freshly prepared before each dose.Additionally, we compared different doses and frequencies of Lodamintreatment: 15 mg/kg q.d., 15 mg/kg q.o.d. and 30 mg/kg q.o.d. Toeliminate any possible effect of the vehicle (polymer without drug) onegroup of mice was given micelles without drug and tumor progression wascompared to water treated mice.

For the melanoma tumor experiment, a dose of 15 mg/kg q.d. Lodamin wasadministered to B16/F10 melanoma bearing mice. In all experiments, tumorsize and animal weight were monitored every 2 d. Tumor volume wasmeasured with calipers in two diameters as follows:(width)×(length)×0.52. Note that all the above Lodamin doses arepresented as TNP-470 equivalent.

Oral Administration of Lodamin and Liver Metastasis Experiments

To examine the effect of oral Lodamin treatment on metastasisdevelopment and prevention, liver metastases were generated by spleeninjection. C57Bl/6J mice (n=14) were anaesthetized with Isoflurane andprepared for surgery. A small abdominal incision was made in the leftflank and the spleen was isolated. B16/F10 tumor cells in suspension (50μl, 5×105 in DMEM medium without serum) were injected into the spleenwith a 30-gauge needle, and the spleen was returned to the abdominalcavity. The wound was closed with stitches and metal clips. After 2 dmice were divided into two groups, one was treated daily with oralLodamin (15 mg/kg) using gavage and the second group was administeredwater by gavage. After 20 d, we terminated the experiment. Mice werekilled and autopsied, livers and spleens were removed by surgicaldissection, imaged, and histology was carried out. Liver and spleentissues were stained with Hematoxylin & Eosin to evaluate tissuemorphology and detect metastasis.

Immunohistochemistry was carried out to specifically detect B16/F10cells in the liver using anti-mouse melanoma antibody (HMB45, abCAM) andusing DAB staining

Evaluation of Neurotoxicity with Balance Beam Motor Coordination Test

A slightly modified balance beam motor coordination test was performedon three groups of mice: Oral Lodamin treated mice (30 mg/kg eq.q.o.d.), free TNP-470 (30 mg/kg) subcutaneously injected mice and watertreated mice (administered by gavage). The mice were pretreated for 14 d(n=4-5 per group) and then the mice were allowed to acclimate to theprocedure room for 1 h, after which they were trained in 3 trials tocross a wide (20 mm width×1 m length) balance beam. All the mice crossedthe wide beam without making foot-slip errors. The mice were thentrained on a narrow (4 mm width×1 m length) beam for 3 trials. At theend of the training trials, no freezing behavior was observed, and themice would start to walk within 4 seconds of being placed on the beam.The mice were then videotaped as they performed in 3 test trials of 3beam crossings each—a total of 9 crossings per mouse. The 3 trials wereseparated by at least 1 h to avoid fatigue of the mice. Videotapedcrossings were scored for number of foot-slip errors and time to cross.All experiments and scoring of the different groups were performed by aninvestigator who has been no knowledge regarding the treatment regime ofthe mice.

MetAP-2 Inhibitor Polymersome Effect on Angiogenesis, Proliferation andApoptosis in Tumor Tissues

Histologic evaluation of tissue was performed on 8 μm thick frozensections of LLC tumors that were removed from two random Lodamin-treatedor untreated mice 14 d post treatment (15 mg/kg q.d. TNP-470equivalent). Tumors were sectioned and analyzed for cell markers, 20microscope fields (×400) were imaged.

Tissues were stained with Hematoxylin & Eosin to detect tissuemorphology. Immunohistochemistry was carried out using Vectastain EliteABC kit (Vector Laboratories, Burlingame, Calif.). Primary antibodiesincluded CD31 (BD Biosciences, San Jose, Calif.) for microvesselstaining and anti-Ki-67 (DAKO, CA, USA) detection of proliferation.Detection was carried out using a 3,3′-diaminobenzidine chromogen, whichresults in a positive brown staining Apoptotic cells were detected byreacting the tissues with Terminal deoxynucleotide transferase mediateddUTP-biotin nick end labeling (TUNEL) using a kit (Roche) following thecompany's protocol. Vessels were detected in the same tissues byanti-CD31 and secondary FITC anti-mouse antibody (JacksonImmunoResearch) conjugated antibody (green) and nuclei were detected byDAPI (blue).

Delayed Type Hypersensitivity (DTH) Reactions

DTH reactions were induced in the skin of 8-week-old C57Bl/6J male miceas previously reviewed⁶².

Induction of CNV and Flat-Mount Preparation

Laser-induced CNV was generated by a technique described previously withsome modifications⁶³. Only lesions in which a subretinal bubble or focalserous detachment of the retina developed were used for the experiments.Lodamin treatments were started at the day of CNV induction and thesizes of CNV lesions were determined after 7 or 14 d. For this purpose,four burns were performed per eye while leaving a space around the opticdisc. After 7 or 14 d of laser induction, the retinas were analyzed forblood vessel formation.

Analysis of Cell Population in Retina by Flow Cytometry

Cell populations in retina tissues were analyzed by FACS.

In-Vivo Miles Assay for Vessel Permeability

Miles assay was performed in C57Bl/6J mice 5 d after oral Lodamintreatment (30 mg/kg/day). MCP-1 and VEGF-165 were used as inducers.

Laser Induced Retinal Vascular Permeability and Biodistribution Studies

In order to evaluate specifically the effect of Lodamin on laser-inducedretinal leakage while excluding the anti-angiogenic activity of Lodamin,CNV lesions of the same size (same day post laser injury) were used tocompare blood vessel permeability in mice and rats. Two different assayswere performed; Angiography in live rats and modified Miles assay inmice. A modified Miles assay was performed as described in the Examples.Angiography was performed on rats using Fluorescein imaging aspreviously described⁶⁴.

In order to evaluate biodistribution of Lodmain in CNV site, fluorescentlabeled drug was used.

Electroretinography

Dark-adapted full-field ERGs were obtained as previously described⁶⁵. Byfitting the Hood and Birch formulation⁶⁶ of the Lamb and Puchmodel^(67, 68) of phototransduction to the a-wave, we obtained thephotoreceptor's kinetics (Srod) and saturated amplitude (RmP3). From theresponse vs. intensity relationship of P2, the putatively-purepostreceptor response derived from the b-wave, we determined bipolarsensitivity (log kP2) and saturated amplitude (RmP2). The oscillatorypotentials (OPs) obtained after passing P2 through a 5th-orderButterworth filter (bandpass 50-250 Hz); were evaluated in the frequencydomain⁶⁹ to estimate maximal OP energy (Em). Finally, the retinalresponse to light-adapted 8 Hz flicker was measured (R8). ERG data wereexpressed as ALogNormal.

Analysis of Cell Population in Retina by Flow Cytometry

Single cell suspensions were prepared from C57BL/6J mouse retinas withCNV lesions. In order to collect a sufficient number ofocular-infiltrating cells for flow cytometry, 20 separate burns weredelivered similar to the panretinal photocoagulation procedure inhumans. The quantity of macrophages in single-cell suspensions frommouse retinas from days 3 and 7 post laser procedure was evaluated forLodamin treated (oral, 30 mg/kg q.d for 3 days) or vehicle or untreatedmice. In the third day of treatment mice were euthanized and analyses oftheir retinas were performed.

Statistical Analysis

In-vitro data are presented as mean±SD, whereas in-vivo data arepresented as mean±SE. Differences between groups were assessed usingunpaired two-tailed Student's t-test, and P<0.05 was considered asstatistically significant.

Example 1

Block copolymer micelles (polymersomes) have been proposed for thedelivery of hydrophobic drugs with low aqueous solubility (such asPaclitaxel). The amphiphilic nature of diblock copolymers such aspoly(lactide)-poly(ethylene glycol) (PLA-PEG), enables the formation ofmicelles in aqueous media. In an aqueous environment, the diblockcopolymer is self-assembled into a structure of hydrophobic core, formedby the association of the hydrophobic polymer and the drug, and ahydrophilic shell, formed by the hydrophilic polymer, commonly PEG.

Block copolymers as disclosed in U.S. Pat. No. 4,745,160 (which isincorporated herein by reference) have been used form water insoluble,amphiphilic, non-crosslinked linear, branched or graft block copolymershaving polyethylene glycol as the hydrophilic component and poly(D-, L-,or D,L-lactic acids) as the hydrophobic components.

Block copolymers as disclosed in U.S. Pat. No. 5,543,158 (which isincorporated herein by reference) have been described to formnanoparticles or microparticles that are solid particles that aresuspended in water that are formed from a water-insoluble blockcopolymer consisting essentially of poly(alkylene glycol) andpoly(lactic acid). The molecular weight of the block copolymer is highand the copolymer is insoluble in water. In the nanoparticle ormicroparticle, the biodegradable moieties of the copolymer are in thecore of the nanoparticle or microparticle and the poly(alkylene glycol)moieties are on the surface of the nanoparticle or microparticle in anamount effective enough to decrease uptake of the nanoparticle ormicroparticle by the reticuloendothelial system. Nanoparticles areprepared by dissolving the block copolymer and drug in an organicsolvent, forming an oil and water emulsion by sonication or stirring,and collecting the nanoparticles containing the drug followingprecipitation. It does not provide for the solubilization of hydrophobicdrugs.

The inventors demonstrate that MetAP-2 inhibitor TNP-470 wassuccessfully conjugated to a modified PEG-PLA polymer through its amine,and formed nano-size polymersomes. A schematic diagram of theconjugation process is shown in FIG. 6A. Step I demonstrates thereaction between succinated mPEG-PLA and ethylendiamine that results inan amine terminated polymer. Step II demonstrates the reaction betweenthe amine reactive polymer and the terminal chlorine of TNP-470. Theconjugate is then dialyzed against water in an excess of TNP-470 to formpolymeric micelles. The inventors demonstrate, using images taken byTEM, that spherical micelles were formed and size measurement with DLSshowed a low range of size distribution around 10 nanometers. By usingconfocal microscopy images of fluorescent-labeled polymersomes, theinventors demonstrate rapid uptake by Human Umbilical Vein EndothelialCells (HUVECs), and when TNP-470 polymersomes were added, a significantinhibition of HUVECs proliferation was shown (as compared to no effectof the carrier itself).

In-vivo studies done on mice showed a significant inhibition ofsubcutaneous Lewis Lung Carcinoma tumors with C57/BL mice given a dailyoral administration of TNP470 micelles. A dose of 15 mg/kg TNP-470equivalent showed 63% inhibition without any weight loss to the mice.

NMR results from the different reaction steps are shown in FIG. 1. Theresults show the binding of the ethylendiamine through the carboxic acidgroup at the original polymer, and the TNP-470 binding to the polymerthrough the amine.

Amine Determination.

To further verify the binding of TNP-470 to the modified PEG-PLA throughthe amine in the polymer and the terminal chlorine in the drug, a TNBSAreagent reaction for the detection of free amino groups was performed.According to the results (FIG. 2) the polymer was saturated withethylendiamine. Correlation between molar amine groups in the modifiedpolymer and absorption in 450 nm were determined by a theoretical valuefrom a standard curve done for aspartic acid (not shown). Moreover,after TNP-470 conjugation, no free amines were detected, indicating thatthe polymer was saturated with TNP-470 conjugated through the aminegroup at the polymer.

Morphology of MetAP-2 Inhibitor Polymeric Micelles by TransmissionElectron Microscopy (TEM).

In order to study the morphology of TNP-470-micelles, TransmissionElectron Microscopy (TEM) images were taken at the day of preparation(data not shown).

Polymersomes dispersed in double distilled water were imaged withcryo-TEM (JEOL 2100 TEM, Harvard university-CNS). The images show thatthe self-assembled polymersomes had a uniform spherical morphology.

HUVEC Proliferation.

The effect of different concentrations of TNP-470 polymersomes on theproliferation of HUVECs was evaluated using a WST-1 reagent. FIG. 4shows the inhibition in proliferation after 48 hr of incubation. FIG. 4Ashows the effect of TNP-470 micelles, and FIG. 4B shows the effect ofmicelles without drug on HUVECs. A significant inhibition of HUVECproliferation was detected from 0.3 mg/ml and up to 2.4 mg/ml micelles,which are 62.5 nM-1000 nM TNP-470 equivalent (88% and 95% inhibitionrespectively). The same concentrations of polymeric micelles withoutTNP-470, as well as double concentration (4.8 mg/ml) showed nosignificant effect on HIJVEC proliferation.

Example 2 Cellular Uptake Studies

In order to evaluate the kinetics of polymersomes uptake by HUVECs inin-vitro condition, HUVECs were incubated with fluorescent polymersomes,washed and imaged by Confocal microscopy. HUVECs were incubated with themicelles for 20 min, 4 hr and 24 hr. After 20 min. micelles were alreadyuptaken by the cells and located at the cytoplasm (data not shown).After 2 hr, 4 hr, 7 hr and 24 hr the uptake increased and the micelleswere detected as concentrated spots inside cell cytoplasm (data notshown).

In Vivo Effects of PEG-PLA-MetAP-2 Inhibitor Micelles.

C57/BL mice bearing s.c LLC tumors showed inhibition in tumor growthwhen treated with PEG-PLA-TNP-470 micelles given orally. All dosesshowed a significant effect already after 14 days of daily treatment, asshown in FIG. 5A. The controlled groups had to be scarified on day 14,and the treatment continued up to day 18. The lowest dose (15 mg/kgTNP-470 equivalent) showed a significant inhibition of tumor growth(63%) after 14 days (*p<0.05), without any weight loss to the mice.whereas higher doses of 30 mg/kg and 60 mg/kg TNP-470 equivalent showed74%, 75% inhibition respectively, but also caused almost 20% weight loss(FIG. 5B).

Absorption of Polymeric Micelles in the Gastrointestinal Tract.

The non-invasive Xenogen system was used to track the absorptionkinetics of the polymersomes after oral administration. The micelleswere concentrated at the gastrointestinal tract within 10 min postadministration (GI track) (data not shown). Apparently the absorptionwas rapid and after 1 hr fluorescent signal was found near the bladderand the prostate glands. After 24 hr the signal was still detectable.

Example 3 Chemical and Physical Characterization of MetAP-2 InhibitorPolymersomes

In an effort to predict the oral availability of TNP-470, thehydrophobicity of Lodamin was measured using log-D parameter. Themeasured Log-D values were 2.39 at pH=2 and 2.57 at pH=7.4. The highLog-D values (>2) indicate a very low solubility in water. This propertyindicate that the designed Lodamin had with improved solubility and oralavailability.

The formulation of Lodamin and the conjugation of TNP-470 are describedin detail in the Methods. The chemical and physical properties ofLodamin was characterized. First, the binding of TNP-470 to mPEG-PLA wasconfirmed by ³H NMR (data not shown) and mass spectrometry showing anaverage m/z of 3687 for mPEG-PLA-TNP-470. FIG. 6A demonstrates thereaction of Lodamin preparation. Using amine detection reagent, theincorporation efficiency of ethylenediamine to mPEG-PLA was determinedas 65%, and in the second step TNP-470 was shown to be bound in highefficiency of >90%. Lodamin contained 0.8-1% (w/w) free TNP-470 asdetermined HPLC. The average size and size distribution of Lodamin wasdetermined by a DLS (FIG. 3) on the day of preparation and after 10 d ofincubation in aqueous medium to evaluate Lodamin stability (n=4). Themajority of micelles (90%) at day of preparation were 7.8-8 nm indiameter, with a small population of larger particles (200-400 nm). Thesize remained almost unchanged after 10 days.

The morphology of Lodamin was characterized by TEM. (FIG. 6B). Theimages showed that the polymeric micelles had acquired a uniformspherical structure, which remained stable after two weeks of incubationin water at 37° C. Since the drug is located in the PLA core of themicelle structure and PLA is spontaneously hydrolyzed in an aqueousenvironment, we studied the release kinetics of TNP-470 from Lodamin. Aslow-release kinetic of TNP-470 was obtained following incubation in PBS(pH=7.4) or in gastric liquid (pH=1.2). The TNP-470 was released over aperiod of 28 d with an early peak burst of ˜30% after the first day ofincubation (FIG. 6C).

Endothelial Cells Take Up MetAP-2 Inhibitor Polymersomes by Endocytosis

The uptake of polymeric micelles by HUVEC and its kinetics was evaluatedinitially. HUVEC were incubated with 6-coumarin labeled mPEG-PLAmicelles for 20 min, 2, 4 and 7 h and were imaged by confocalmicroscopy. Confocal images show HUVEC uptake of polymeric micelleslabeled with 6-coumarin after 20 min and 7 h incubation time periods(data not shown). In Live HUVEC cells as were imaged 1 h post theaddition of labeled micelles to cell media (15 μg/ml, in green).LysoTracker Red® was used to detect Endosomes and Lysosomes. Overlaybetween micelles and Endo-lysosomes is represented in yellow/orangecolor (data not shown). As soon as 20 min after incubation, micelleswere taken up by the cells and was located in their cytoplasm. After 2 hthe uptake was maximal and after 4 to 7 h micelles were detected asdefined aggregates inside the cytoplasm. Flow cytometry analysis ofHUVEC incubated with rhodamine-labeled polymeric micelles for the sameincubation times confirmed a maximal uptake after 2 h (FIG. 7A), while,no difference was observed between 2 to 24 h of incubation. In live-cellanalysis, co-localization of Lyso-tracker staining with the micellessuggests endocytosis mechanism of uptake. Incubation of the micelleswith HUVEC in cold conditions reduced micelle's uptake by up to 55%,confirming the endocytosis process (data not shown).

MetAP-2 Inhibitor Polymersomes Inhibit Proliferation of EndothelialCells

Following polymeric-micelle uptake studies, the effect of Lodamin on theproliferation of endothelial cells was evaluated. Lodamin (62.5 nM-1,000nM TNP-470 equivalent) inhibited HUVEC proliferation by 88% to 95%respectively after 48 h (FIG. 7B). The growth of HUVEC treated withLodamin (60 nM TNP-470 equivalent) was completely inhibited compared tountreated cells or cells treated by vehicle only. No substantialcytotoxic effect was found (FIG. 7C).

MetAP-2 Inhibitor Polymersomes Inhibit VEGF and bFGF-InducedAngiogenesis In Vivo

The anti-proliferative and antiangiogenic properties of Lodamin wereevaluated in-vivo by the corneal micropocket assay²⁹. Mice were treatedwith daily oral Lodamin (15 mg/kg q.d.) or vehicle for 6 d. Newly formedblood vessels grew towards the bFGF pellet (data not shown) inrepresentative eyes of untreated mice. Treated mice show the inhibitionof bFGF induced angiogenesis in representative eyes. Quantification ofthe proliferative area (FIG. 7D) showed 31% inhibition of angiogenesis,compared to vehicle (P=0.00016, n=10). Similar results were obtainedwith VEGF165 (160 ng) induced angiogenesis in the cornea (data notshown), in this case Lodamin resulted in 40% inhibition of vessel area.

Polymeric Micelles: Absorption by the Intestine

To study the intestinal absorption of mPEG-PLA-rhodamine micelles, micewere administered oral polymeric-micelles. After 2 h mice wereeuthanized and isolated segments of the small intestine were fixed andimaged using confocal microscopy. A histological section of gutepithelial cells of Lodamin treated mouse was observed with TEM. Thepolymeric micelles were intensively taken up by columnar epitheliumlining the luminal side of the small intestine. In a high magnificationof small intestine villi, micelles are clearly detected in the laminapropria and in the vicinity of the blood vessels, indicatingtransepithelial absorption. In high resolution TEM images of single gutepithelial cells, many endosomes loaded with the drug were detected(data not shown). Microvilli (MV) structures and endosomes loaded withpolymeric micelles (data not shown) are detected. Bar=500 nm (left) and200 nm (right). Confocal microscopy image also confirms the TEMobservations. Note the polymeric micelles can be detected in the laminapropria in the vicinity of blood vessels. The actin filaments werestained with phalloidin-FITC, nuclei were labeled with DAPI and mPEG-PLAwere labeled with rhodamine, bars=5 μm. These vesicles were different incontrast and number than in an intestine of untreated mouse. These datasupport the premise that Lodamin was absorbed by the villous structureof the intestine by endocytosis.

Body Distribution of Polymeric-Micelles, Accumulations in Tumors andToxicity

The biodistribution and tissue uptake of orally administered labeledMetAP-2 inhibitor polymersomes were studied by treating mice withfluorescent labeled PEG-PLA micelles for 3 d. After harvesting thetissue, tissue drug concentration was quantified by dye extraction or byflow cytometry. The results of the fluorescent dye extraction method(FIG. 8A) showed that as expected, a high concentration of fluorescentsignal was detected in the stomach and the small intestine, where theliver showed the highest level. Importantly, the brain showed nopresence of fluorescent signal. In the serum, labeled micelles werealready detected after 1 h post oral administration, peaking after 2 hand were still detected up to 72 h. Accordingly, in tumor bearing mice,flow cytometry analysis of enzymatically digested tissues (FIGS. 8C andD) demonstrated a large uptake of labeled micelles by the liver, and nouptake by brain cells. Importantly, the highest uptake of micelles wasdetected in tumor cells. In FIG. 8C the percentage of FL2^(high) cells,i.e. cells that absorbed rhodamine-labeled micelles is demonstrated.Taken together, these results indicate that the drug is mostlyconcentrated in the tumor and in the gastrointestinal organs (althoughto a lower extent) and not in the brain.

No tissue abnormalities were detected in histological analysis (H&E) ofliver, intestine, lung and kidneys of MetAP-2 inhibitor polymersometreated mice (15 mg/kg TNP-470 equivalent q.d., for 20 d) when comparedto untreated mice (data not shown). In addition, no significantdifferences were found in mouse serum liver-enzyme profile of MetAP-2inhibitor polymersome treated mice compared to untreated mice. InLodamin treated group ALT concentration was 41±9 u/l and AST was 120±39u/l whereas in the untreated group the ALT concentration was 37.5±4 u/land AST concentration was 152±131 u/l.

MetAP-2 Inhibitor Polymersomes Inhibit Primary Tumor Growth

The biological efficacy of Lodamin was evaluated as an anti-cancer agentin tumor bearing mice. When mice were given oral free TNP-470 at a doseof (30 mg/kg q.o.d.) no inhibition in the growth of subcutaneous LLCtumors was observe (FIG. 9A). The equivalent dose of Lodamin, however,resulted in a significant inhibition of tumor growth (FIG. 9A). Thisinhibition was observed after 12 d of Lodamin treatment, and at day 18tumor growth was inhibited by 83%. Different dosing strategies ofLodamin were tested: 15 mg/kg q.d., 30 mg/kg q.o.d. and 15 mg/kg q.o.d.,resulting 87%, 77%, and 74% of tumor volume inhibition respectively(FIG. 9B). The vehicle (PEG-PLA) showed no effect on tumor growth andwas similar to untreated control mice (FIG. 9C).

In another tumor model, B16/F10 melanoma tumors were inducedsubcutaneously and their growth was also inhibited by oral Lodamin (15mg/kg q.d.). This treatment was effective as of day 4, and after 13 d77% volume inhibition was obtained (FIG. 9D). No apparent side-effectsor weight loss were detected in either tumor models. Higher doses ofLodamin: 30 mg/kg q.d., and 60 mg/kg. q.o.d., showed substantial tumorinhibition, however, it was accompanied by weight loss (data not shown).FIG. 9E shows representative tumors of treated or untreated LLC orB16/F10 tumors.

MetAP-2 Inhibitor Polymersomes Did not Cause Neurotoxicity in Mice

Since the biodistribution study indicated that Lodamin does not crossthe blood brain barrier, we further tested if the possible penetrationof escaped free drug into the brain might still result in neurotoxictyand cerebellar dysfunction. To examine this, mice were subjected to asensitive test of motor coordination: crossing a narrow (4 mm) balancebeam30. As shown in FIG. 9F Lodamin-treated mice (30 mg/kg q.o.d. for 14d) performed in this challenge similar to control (water treated) mice,whereas mice injected with free TNP-470 (30 mg/kg q.o.d.) committed overtwice as many errors (P<0.0001). These results indicate that Lodamintreatment avoids the cerebellar neurotoxicity observed with unconjugatedTNP-470 treatment.

Oral MetAP-2 Inhibitor Polymersomes Inhibit Angiogenesis and CellProliferation in Tumors and Induce Tumor Cell Apoptosis

The effect of Lodamin on the histological structure of the LLC tumorswas tested. Both treated and untreated tumors showed a dense cellularstructure (data not shown). Lewis lung carcinoma (LLC) tumors wereremoved from Lodamin treated or untreated mice and sectioned. Tissueswere stained with Hematoxylin & Eosin (H&E) to detect tissue morphology.Immunostaining with anti-CD31 was used to detect microvessels andanti-Ki67 nuclear antigen for cell proliferation. TUNEL staining wasused for the detection of apoptotic cells. Cell nuclei were stained withDAPI. Sections were counterstained with Eosin (nuclei). The tumors ofuntreated mice had a net organization of small and large vessels withapparent lumen structure as demonstrated by CD-31 immunostaining. Incontrast, Lodamin treated tumors formed very small undeveloped vessels(data not shown). Lodamin treated tumors showed less cellularproliferation than untreated tumors, as detected by the nuclear markerKI-67. TUNEL staining for the detection of apoptosis in the tissuesindicated an enhanced apoptosis in Lodamin treated tumors. Lodamintreated tumor had less vessels but high levels of apoptosispredominantly of tumor cells. In the control tissue, apoptotic cellswere found in the capsule of the tumor but less in the center of thetissue.

In addition, the new oral formulation of the MetAP-2 inhibitor TNP-470did not cause any weight loss or other apparent side-effects in the mice(FIG. 9G).

MetAP-2 Inhibitor Polymersomes Prevent Development of Liver Metastasis

The effect of oral Lodamin administration on liver metastases was testedafter spleen injection of B16/F10. Oral Lodamin treatment dramaticallyaffected development of liver metastasis. After 18 d of treatment micewere autopsied. All untreated mice had ascites while their enlargedlivers had macroscopic malignant nodules and extensive cirrhosis, (FIG.10A). In contrast, Lodamin-treated mice had no macroscopic metastasis inthe abdomen and in the liver (FIG. 10A). Their organs had normalmorphology and no weight-loss or other apparent side-effects were found.Immunohistology showed only a few sporadic B16/F10 cells in the liver,which had not developed into lesions (data not shown). Only one treatedmouse out of seven had malignant nodules in its liver, but the liver wassmaller than in the untreated control and had less cirrhosis. Thespleens of all Lodamin treated mice had normal morphology compared tothe congestion found in the enlarged spleens of control mice (FIG. 10B).Twenty days post B16/F10 cell injection into the spleen, 4 out of 7control mice had died while all treated mice survived (FIG. 10C).

In this study, the inventors describe for the first time the developmentof a nontoxic oral formulation of the MetAP-2 inhibitor TNP-470, namedLodamin, as a potent anti-proliferative and anti-metastatic drug.TNP-470, a highly potent anti-proliferative/antiangiogenesis agent, is aleading candidate for chronic administration for cancer maintenancetherapy and metastasis prevention.

The challenge that the inventors faced was based on the fact thatTNP-470 has very poor oral availability as illustrated by its high Log Dvalues, indicating low water solubility³⁵. In order to make the drugsuitable for oral administration, this property of TNP-470 was alteredwhile retaining its activity by conjugating it with mPEG-PLA to formmPEG-PLA-TNP-470 polymeric micelles i.e. Lodamin. Unlike TNP-470 whichis only dissolvable in organic solvents, Lodamin can be suspended inwater to form polymeric micelles caused by the amphiphilic nature ofPEG-PLA²⁶. In this structure the drug is located in the core of themicelle protected from the harsh gastrointestinal environment³⁶. Polymermicelles have previously been used for the delivery of hydrophobicdrugs^(37, 38) and gene delivery³⁹. Lodamin acquired a stable sphericalmorphology of nanomicelles, as imaged by TEM. In addition, PLA which isa biodegradable and biocompatible polymer, hydrolyzes in an aqueousenvironment and allows a slow-release of TNP-470. In-vitro studiesshowed a continuous release of TNP-470 from Lodamin over almost a monthperiod, where the majority of the drug was released after 4 d (in bothgastric and plasma pH conditions), although acidic environment is knownto accelerate degradation of PLA we observed only a minor effect on day15. While not wishing to be bound by theory, one possible explanationmay be the masking effect of the PEG shell which delays the waterpenetration to the PLA core and slows the release of the drug throughdiffusion of this layer. In culture, Lodamin was rapidly taken up byendothelial cells via endocytosis and retained the originalantiangiogenic activity of the free TNP-470 as demonstrated by theinhibition of HUVEC proliferation and growth rate.

PEG-PLA micelles penetrated the gut epithelial layer into the submucosaas shown by using fluorescent marker. The mechanism of Lodaminpenetration to gut epithelial cells seems to be via endocytosis asdetected by high resolution TEM imaging. In serum, labeled micelles hadlong blood circulation time of at least 72 h post administration, asignificant increase compared to free drug which was detected in micesera up to 2 h post-administration. Biodistribution showed relativelyhigh concentration in the liver because oral administration delivers thedrug directly from the intestine to the liver. However, no livertoxicity was observed by histology and by liver enzymes profile after 20d of daily Lodamin treatment.

Lodamin given orally to mice showed substantial anti-proliferativeeffects (83% reduction), while free TNP-470 given orally had no effect.The effect on LLC growth was dose dependent, as 30 mg/kg q.o.d. was moreeffective than 15 mg/kg. q.o.d., and a dose of daily 15 mg/kg q.d. wasmore effective than 30 mg/kg q.o.d. (a double dose given every 2 d). Asimilar anti-proliferative effect of Lodamin was also observed withmelanoma B16/F10 tumors, confirming the broad biological effect ofLodamin, very much like the original free TNP-470. Immunohistochemicalstudies carried out on LLC tumor tissues showed a reduction ofproliferation and angiogenesis induced by Lodamin. Using TUNEL stainingwe detected a high level of tumor cell apoptosis following the Lodamintreatment, whereas in the controls much of the apoptosis occurred on thecapsule of the tumor. These results indicate that the prevention ofangiogenesis by Lodamin leads to tumor cell apoptosis, thus makingLodamin a cytotoxic drug, in addition to an antiangiogenic drug.

One of the most notable effects of oral Lodamin is the prevention ofliver metastasis in mice. Liver metastasis is very common in many tumortypes and has often been associated with poor prognosis and survivalrate. The inventors chose the intrasplenic model for induction of livermetastasis, in which the transition time of B16/F10 from spleen to livermicrovasculature post spleen injection in mice was found to be very fast(20% of the injected cells after 15 min) 40. Mice injected with B16/F10cells into the spleen had a low survival rate. They developed ascites,macroscopic malignant nodules, and extensive cirrhosis in their livers20 d post injection. However, all oral Lodamin treated mice survived upto this point and had normal liver (except one mouse) and spleenmorphology without any other apparent side-effects. Immunohistology oftreated mice's livers showed some rare sporadic B16/F10 cells whichremained dormant over the 18 d of oral Lodamin treatment, compared tolarge metastasic tumors found in the untreated mice. It should be notedthat Lodamin's effect on secondary tumor growth is in addition to itseffect on the primary tumor.

While not wishing to be bound by theory, this dramatic effect may be dueto the oral route of administration in which Lodamin is absorbed in thegastrointestinal tract and concentrated in large quantities in the livervia the portal vein. These results indicate that Lodamin can prevent thedevelopment of metastasis in the liver associated with different tumortypes. Importantly, this property of the polymeric micelles might beused for enabling oral availability of other antiangiogenic oranti-cancer drugs to target liver metastasis.

In summary, oral Lodamin is therapeutically effective in the treatmentof solid tumors and metastasis in mice. It captures theantiproliferative properties of free TNP-470 while adding importantadvantages: oral availability, improved biodistribution, tumoraccumulation, continual release, improved solubility, proper clearance,and low related side-effects. In addition to use for the treatment orsuppression of known, existing tumors, its use is contemplated, amongothers, for cancer patients as a long-term maintenance angiogenesisinhibitor to prevent tumor recurrence. Furthermore, it is alsocontemplated for use as maintenance therapy for chronic MetAP-2-relateddiseases such as age-related macular degeneration, endometriosis andrheumatoid arthritis. Other contemplated uses are described elsewhereherein.

Example 4 MetAP-2 Inhibitor Polymersomes Inhibit Glioblastoma TumorGrowth

The effect of Lodamin on human brain tumor (U87MG, Glioblastoma) wasstudied using subcutaneous tumor-bearing nude mice. Treatment withLodamin started when tumors were in volume of ˜100 mm³. Lodamin that wasgiven by gavage (FIG. 11A) in a daily dose of 15 mg/kg TNP-470equivalent, inhibited tumor volume by 70% after 30 days. When Lodaminwas given in drinking water at the same dose (FIG. 11B) a 71% inhibitionof tumor volume was obtained after 42 days.

Example 5 MetAP-2 Inhibitor Polymersomes Prolong Survival of MiceBearing Lung Metastasis

C57/Bl mice were injected into their tail-veins with 2.5×105 B16/F10cells. Day after injection the mice were divided into 3 groups: 10 micewere given a daily oral administration of Lodamin (15 mg/kg TNP-470equivalent), 9 mice were given IP injection with the same Lodamin dose,and 10 mice remained untreated. Mice survival was followed up to 40 dayspost treatment.

Lodamin treated mice showed a prolonged survival as can be seen inKaplan-Meier survival curve (FIG. 12). After 32 days all control micedied, compared to 6 and 5 mice that lived at day 40 in the IP Lodamintreated group and Oral Lodamin group respectively.

Example 6 MetAP-2 Inhibitor Polymersomes Inhibit Angiogenesis inMatrigel Angiogenesis

In-Vivo Matrigel Angiogenesis Assay was conducted in order to assess theantiangiogenic properties of Lodamin. Matrigel (BD bioscience) which wasremained in 4° C. overnight was mixed with fibroblast growth factor-2(FGF-2) to obtain a final FGF-2 concentration of 1 μg/ml. Nine C57/Blmice were injected with Matrigel (500 μl) in two sites subcutaneously.Three mice got a daily oral treatment with Lodamin (15 mk/kg/day TNP-470equivalent). In three mice the Matrigel was mixed with Lodamin (5 mg/ml)and no further treatment was performed and three untreated mice withMatrigel implant were used as a control.

After 8 days, mice were euthanized and the Matrigel implant harvested,washed twice with PBS, and then one of the implant was immediatelyfrozen on dry ice and used for histology assessment of blood vesselsusing CD-31 antibody. The second implant was enzymaticaly digested usingcollagenase (Librease, Roche) to obtain single cell suspension whichagain was analyzed by FACS to quantify the number of endothelial cellsusing CD-31 and CD-45 antibodies (endothelial cells are CD31+CD45−).

The implants of Lodamin treated mice, both the one with the mixture ofmatrigel and Lodamin or the mice treated orally with Lodamin, showed asignificant reduction in blood vessels inside the implants (FIG. 13A).Histology showed less vessels in Lodamin treated mice and less densetissue. FACS analysis suggests 70% reduction in the percent ofendothelial cells which were attracted to the implant (FIG. 13B).

Example 7 Broad Spectrum Antiangiogenic Treatment for Ocular NeovascularDiseases

Introduction

Ocular neovascular-related diseases are associated with visionimpairment or vision loss in millions worldwide. The growth of abnormal,leaky blood vessels is a prominent component of several debilitating eyediseases including AMD, proliferative diabetic retinopathy (PDR), andretinopathy of prematurity (ROP). Thus antiangiogenic strategies couldbe particularly beneficial in preventing and treating the progression ofthese diseases. Corneal neovascularization is often the result ofinflammation, chemical burns, and conditions related tohypoxia^(48, 49). These conditions are currently treated by indirectangiogenesis inhibitors such as steroids and immunosuppressants⁴⁸.Efforts to inhibit retinal neovascular disease such as ARMD have focusedon the angiogenic factor Vascular Endothelial Growth Factor (VEGF) andsuch therapies are now FDA approved. The most prominent class ofanti-VEGF therapy is represented by ranibizumab and bevacizumab. Thesemolecules are derived from a humanized anti-VEGF antibody which has theability to reduce the retinal edema in ARMD⁵⁰. However, the effects ofthese therapies are short lived and thus repeated intravitrealinjections every 4 weeks is necessary⁵¹. While clinical trials haveshown a beneficial effect in 30-40% of treated patients^(50, 51), inmany cases these therapies become less effective over time⁵² and thelong-term safety is still debated.

In the recent years it has become clear that diverse cellular processeshave been implicated in pathogenesis of retinal diseases, such as ARMDor PDR. These processes include oxidative stress, vascular permeability,fibrosis, inflammation and impaired function of RPE⁵³. Given themultifactorial nature of neovascularization and the contribution of themicroenvironment, the focus on more effective therapies to block CNV hasshifted toward targeting multiple processes rather than a singlepathway⁵⁴.

In this study it was demonstrated that a broad spectrum antiangiogenicdrug can be especially beneficial in inhibiting CNV by blocking vesselgrowth, reducing vascular leakage and modifying the neovascular stromavia suppression of inflammation. This type of therapy thus offers animprovement over current treatments and an alternative where existingtherapies fail.

Lodamin is a novel polymeric antiangiogenic drug and the first oralformulation for TNP-470⁵⁵. Lodamin is a conjugate of TNP-470 andmonomethoxy polyethyleneglycol-polylactic acid (mPEG-PLA) polymer thatforms a micelle-like structure when introduced in aqueous solution. Theactive molecule, TNP-470, is a synthetic analogue of fumagillin and amethionine aminopeptidase-2 (MetAP2) inhibitor. The broad spectrumactivity of TNP-470 was demonstrated in more than a hundred preclinicalanimal tumor studies. In clinical trials, some cancer patientsdemonstrated partial or complete responses^(56, 57). However, TNP-470also demonstrated poor pharmaceutical properties and dose-limitingreversible cerebellar side effects⁵⁸. Therefore Lodamin was rationallydesigned to overcome these limitations. It acquires superior propertiessuch as: water solubility, improved oral availability, prolonged halflife, slow-release properties and minimal TNP-470-related cerebellarside effects⁵⁵. Lodamin had potent antiangiogenic effects in murinetumor and metastases models.

Using the CNV model in mice and rats, the inventors show that Lodamin isa first-in-its-class polymeric broad-spectrum antiangiogenic ophthalmicdrug that targets numerous molecular pathways in addition to VEGF.Lodamin's unique chemical properties, combined with its mechanism ofaction, place it as a leading candidate for treating corneal and retinalneovascular diseases.

Results

Lodamin Inhibits Angiogenesis and Macrophage Recruitment In Vivo

To investigate the effect of Lodamin on angiogenesis and macrophagerecruitment, the mouse Matrigel angiogenesis assay was first performed.Angiogenesis in the Matrigel was visually assessed after removal of theplugs 7 days post Matrigel injection (FIGS. 16A and 16G) and byimmunohistochemical staining of blood vessels using CD31 antibodies(FIG. 16A). Immunohistology revealed that Lodamin dramatically reducedangiogenesis compared with untreated mice which presented numerous largeblood vessels with open lumen structures (n=5). Infiltrated endothelialcells and macrophages in Matrigel matrices were quantified in asingle-cell suspension by fluorescence-activated cell sorting (FACS)(FIG. 16C). After 7 days of Lodamin treatment, significant reductions ofinfiltrating endothelial cells and macrophages were seen in the Lodamintreated group compared to vehicle treated mice (endothelial cells:0.11±0.06% versus 0.49±0.11%; macrophages: 30±7% versus 43±9% in Lodaminand control groups, respectively).

FIG. 16H shows the immunohistological analysis of mice ears post DTHreaction: staining of macrophages using F4/80 marker. Control earsexhibited an excessive inflammation. Bar=50 μm. No obvious differencewas found in macrophage distribution; however, the total number ofmacrophages present in control mice was elevated due to greater amountof tissue associated with increased swelling.

Systemic and Local Lodamin Treatment Inhibit Corneal Neovascularization

Antiangiogenic activity of Lodamin following topical or systemicadministration was evaluated in the mouse corneal micropocket assayusing bFGF-induced neovascularization. In Lodamin treated mice, cornealvessel area was reduced by 38% relative to the control group aftersubconjunctional injection (30 mg/ml q.d for 5 days), 30% after oncedaily eye drops (30 mg/ml q.d for 5 days) and 37% after oraladministration (30 mg/kg/d q.d for 5 days). FIG. 17B showsrepresentative images taken from the different mouse groups.

Lodamin Reduces Time Course of Delayed-Type Hypersensitivity DTHEar-Swelling

Lodamin's effect on angiogenesis, inflammation, vascularhyperpermeability and edema were evaluated in the DTH reaction. C57Bl/6Jmice were sensitized with oxazolone and treated with Lodamin orally (15mg/kg q.d.) starting from 5 days after the initial exposure tooxazolone. A significant reduction of ear erythema and swelling wasobtained in Lodamin-treated mice compared with vehicle treated mice(FIGS. 16D & 16F). Histology sections stained by Hematoxillin and Eosin(H&E) confirmed a markedly reduced swelling and edema in treated micecompared to untreated mice that presented thick epidermis with extensiveedema demonstrated by cell spongiosis, high cellularity and infiltratingmonocytes (FIG. 16D). A chronic response consisting of neutrophilaccumulation and enhanced fibroblast proliferation was also detected inthe untreated tissues. In some samples, acute inflammation associatedwith focal polynuclear cell accumulations was found.Immunohistochemistry of blood vessels (CD31) revealed a significantreduction of angiogenesis by Lodamin. Some reduction of infiltratedleukocytes and macrophages was observed in Lodamin treated mouse earsstained with CD45 and F4/80 (data not shown).

Oral and Intravitreal Treatment of Lodamin Reduces CNV Progression

The effect of Lodamin on CNV progression in mice was determined afteroral or intravitreal administrations. Oral Lodamin was administered indifferent doses and duration: 15 mg/kg/day (for 7 days or 14 d oftreatment) and 30 mg/kg dose (7 days). CNV lesions in choroidalflat-mounts were evaluated after staining for blood vessels. CNV areawas significantly reduced by Lodamin treatment in all treatment groups.Higher doses and longer treatment led to an enhanced effect: 14 daytreatment (15 mg/kg q.d.) led to 70.6% inhibition whereas 7 day oftreatment at the same concentration led to a 27.2% inhibition. At 30mg/kg/day a more dramatic inhibition was obtained (50.5%) after 7 day.FIG. 18A summarizes the CNV area of all treatment groups. H&E stainingrevealed that Lodamin substantially reduced the formation offibrovascular response from the choroid induced by the spot rupture ofBruch's membrane by the laser (FIG. 18B). Recruitment of macrophagesinto CNV lesions over time was evaluated by FACS in single-cellsuspension originated from retina tissues following Lodamin treatment(30 mg/kg/day, oral). Retinal infiltrated macrophages were reduced afterLodamin treatment in mice (control group: 2% and 3.6%, Lodamin treatedgroup: 1.8% and 2.3%, on day 3 and 7 respectively) (FIG. 18C). WhenLodamin was introduced by a single intravitreal injection using 100 μgand 300 μg Lodamin, a 56% or 75% suppression was obtained respectivelywhen measured after 14 days. No significant difference was found wheninjecting PBS compared to vehicle (data not shown). FIG. 19 showsrepresentative CNV lesions from the different groups and their sizemeasurements. No toxicity was observed in histological H&E whole eyesamples (data not shown).

Lodamin has Minimal Adverse Effect on Retinal Function

To further evaluate possible adverse side effects of Lodamin treatment,retinal function was assessed by electroretinography (ERG) after Lodamintreatment. Normal C57BL/6J mice were injected intravitreal with thehighest dose studied of Lodamin (300 μg; n=10) or vehicle (n=10) andwere tested 14 days after injection. Responses to an ˜6.6 log unit rangeof intensities of full-field flashes presented to the dark-adapted eyewere recorded (FIG. 19B) as was the response to an 8 Hz flickering lightpresented in the presence of an adapting background (FIG. 19C). Rodphotoreceptor response sensitivity (Srod) and saturating amplitude(RmP3) were derived from the ERG a-wave and postreceptor sensitivity(kP2) and saturating amplitude (RmP2) were derived from the ERG b-wave(FIG. 19D). The saturating energy in the ERG oscillatory potentials(Em), and the trough-to-peak amplitude of the flicker response (R8) werealso determined. For each of the six ERG parameters, in log scale, themean and 5th and 95th prediction limits for normal were calculated andcompared to those of Lodamin-treated mice (FIG. 19D); Nearly everyobservation (56 of 60) in Lodamin-treated mice fell within the normalrange. Only RmP3 differed significantly (P=0.46) between groups, by<0.15 log unit. These ERG data confirm the safety of Lodamin withrespect to retina function. Lodamin drug function was further confirmedby histological anaylsis (FIG. 19E). No apparent retinal tissue changeswere detected, both eyes presented normal structures of (1) choroid (2)photoreceptors (3) outer nuclear layer (4) outer plexiform layer (5)inner nuclear layer (6) inner plexiform layer and (7) ganglion celllayer. The total retinal thickness remained unchanged.

Lodamin Reduces Pro-Angiogenic and Inflammatory Factors in CNV BearingMice

Levels of different pro-inflammatory and pro-angiogenic factors weremeasured using specific antibody by enzyme-linked immunosorbent assay(ELISA) or western blot analysis. Significant reductions of MCP-1 andVEGF in retina-choroids tissues were found in Lodamin treated mice byELISA. MCP-1 was not detectable in naïve mice but was found in highconcentrations in CNV bearing mice: 486±3 pg/mg in control CNV mice and61% lower level in Lodamin treated mice (FIG. 20A). Unlike MCP-1, VEGFbaseline levels in naïve mouse eyes were almost as high as in CNVbearing mouse (313 pg/mg, 320 pg/mg respectively) compared to Lodamintreated mice that reduced VEGF levels 24% below the baseline (243pg/mg). MCP-1 levels in primary RPE cultures were also measured by ELISAand was found to be reduced by Lodamin in a dose dependant manner after24 h treatment (39%, 55%, 83% reduction was obtained with 1, 10, 100 nMLodamin, respectively). All ELISA data were normalized to total proteinin each sample (FIG. 20B).

MMP-2 and 9 activities in CNV lesions were determined following oralLodamin treatment (30 mg/kg q.d for 7 d) using zymograms (FIG. 20D). Theenzymatic activity was accelerated in tissues with CNV compared to naïvemouse tissues whereas both MMP-2 and 9 was reduced by Lodamin to thelevels of naïve mice. The levels of TNFα, a major inflammatory cytokine,were higher in choroids than in retina and were reduced in the choroidby Lodamin (FIG. 20C). NF-kB, a pro-inflammatory transcription factorinduced by TNFα, was detected in both retina and choroid tissues and wasalso reduced in the choroid by Lodamin. Lodamin reducedmitogen-activated kinases (MAPK) and its phosphorylated form in thechoroid with no effect in the retina (FIG. 20C).

A dramatic reduction of RAGE levels (35 kDa isoform) were detected inboth choroids and retinas of Lodamin treated mice. HMGB1 (RAGE ligand)was detected in the retina (29 kDa) but no difference was found betweenthe groups. A ˜50 kDa band, possibly corresponding to a multiproteincomplex, was detected in the choroid and was reduced by Lodamin. Invitro RAGE and HMGB-1 levels in microvascular endothelial cells (HMVEC)were reduced in a dose dependent manner by TNP-470 treatment (0.1-100nM) (FIG. 20E).

Lodamin Reduces MCP-1 and VEGF Induced Vessel Permeability

The investigators further investigated whether MCP-1 has a role inLodamin's effect on vessel permeability. In the in vivo Miles assay,MCP-1 enhanced vessel permeability at concentrations of 10 pg/ml (n=15,FIG. 21B). Representative mouse skin patches showing Evans blue leak inMCP-1 injection spots are presented in FIG. 21B. The effect of Lodaminon MCP-1-induced vessel leakage was measured after 5 d of oral treatmentwith 30 mg/kg/day in C57Bl/6 mice. Lodamin significantly reduced vesselpermeability by 87%. Similarly, a standard Miles assay was performedwith VEGF demonstrating 65% reduction of vascular leakage after the sameoral treatment of 5 d.

Lodamin Reduces Ocular Vascular Permeability

In order to evaluate the effect of Lodamin on ocular vascular leak whileexcluding direct angiogenesis inhibitory effects, CNV lesions of thesame stage were used to compare vessel leakage in mice and in rats.Laser-induced CNV in rat retinas were imaged by fluorescein angiography(n=6). Rats with established CNV lesions (7 days after laser injury)were treated with Lodamin once 3 h before measuring the leakage (60mg/kg). The lesions were graded by levels of leakage in laser spots andshowed a significant reduction of retinal vessel permeability by Lodamin(data not shown). A modified Miles assay was also performed to evaluateLodamin's effect on ocular vessel permeability in mice with anestablished CNV lesion. Similarly, when Lodamin (60 mg/kg, n=5) wasadministered once 3 h before Evans blue injection, 65% reduction invessel leakage was obtained by this short-term Lodamin treatment (FIG.21C).

Polymeric Micelles Accumulate in CNV Site

The biodistribution of fluorescent-labeled Lodamin in CNV lesions wasdetermined 3 h post tail vein injection in C57Bl/6J mice. A modelfluorescent hydrophobic small molecule (6-coumarin, MW=350 gr/mole,Sigma) was used to determine differences between accumulations of thefree molecule and its encapsulated form. Blood vessels stained withrhodamine concanavalin A are found in the periphery of the lesion.6-coumarin dye is detected in the center of the lesion that is devoid ofblood vessels. Compared with the free molecule, labeled Lodamin wasdetected in greater extent in CNV sites confirming a passive targeting(n=3) (data not shown).

Many small-molecule angiogenic inhibitors, such as TNP-470, have poorwater solubility necessitating the development of unique formulations tofacilitate their use. In general, particulate drugs offer improvedocular drug delivery since they are usually more stable than othercolloidal systems. Lodamin comprises many of the requirements for anadvanced ocular delivery: it can be introduced into an aqueous solution,is nontoxic, biocompatible, biodegradable, and has slow-releaseproperties⁵⁵. This is the first report of Lodamin treatment for retinalneovascular disorders.

RPE cells play a central role in the progression of AMD disease. Thesecells are responsible for the generation and maintenance of theextracellular and photoreceptor matrices, as well as for the integrityof Bruch's membrane. Impaired function of RPE, occurs in ARMD and causeslipid and protein accumulation in the vicinity of Bruch's membrane andformation of drusen⁷⁰. Furthermore, RPE cells secrete chemoattractantand inflammatory cytokines such as interleukin-8 (IL-8), MCP-1, andTNFα. As a result, neutrophils and macrophages are recruited andstimulate angiogenesis which contributes to ARMD progression⁷¹.

The data demonstrate the suppressive effects of Lodamin on vascularleakage and inflammation, in addition to direct antiangiogenic effects.This was found to be partly due to the substantial reduction of MCP-1production in CNV lesions and in RPE cells. MCP-1 is a small cytokine ofthe C—C chemokine family and is a potent chemoattractant for neutrophilsand macrophages with proangiogenic properties. MCP-1 has been shown tobe elevated in the vitreous of patients with retinalneovascularization^(71, 72) and to be involved in other systemicinflammatory and autoimmune diseases such as diabetes, rheumatoidarthritis and multiple sclerosis^(73, 74). The data show that thesuppression of MCP-1 in the CNV site by Lodamin was accompanied by areduction of infiltrating macrophages. This is very important for ARMDtreatment since macrophages express TNFα in CNV which stimulates furthersecretion of MCP-1, IL-8, and TNFα from RPE cells, thus leading to afeedback loop with additional recruitment of macrophages⁷⁵ and ARMDprogression^(71, 76).

The Miles assay data demonstrated that MCP-1 induced vascular leak in adose dependant manner. Surprisingly, Lodmain was able to antagonizeMCP-1 mediated vascular leak, similar to its antagonism of VEGF inducedleakage. These results provide an explanation for the significantreduction of edema demonstrated in DTH reaction, and of the reduction inretinal edema as analyzed by angiography and Evans blue extraction.Moreover, the reduction of retinal edema by Lodamin was very rapid,where only one pretreatment with oral Lodamin 3 h before performing theassay was sufficient to substantially reduce vessel permeability.

Lodamin effects were not limited to the suppression of MCP-1. Otherpro-angiogenesis and pro-inflammatory factors, including VEGF, nuclearfactor k-B (NFκB), TNFα and matrix metalloproteinases (MMPs), were alsosuppressed in situ by Lodamin treatment. Since NFκB regulates MCP-1 andis involved in the immune response⁷⁷ stimulated by TNF-α and IL-6,suppressing this pathway can potentially lead to a substantial reductionin inflammation and angiogenesis and thereby prevent the progression ofocular neovascular diseases.

It is shown here for the first time that TNP-470 is linked to the RAGEpathway, which plays a pivotal role in retinal diseases such as diabeticretinopathy and AMD^(78, 79-81). RAGE, a multiligand receptor of theimmunoglobulin superfamily, and its ligands: advanced glycolyzationproteins (AGE), high-mobility group box 1 (HMGB-1) and 5100 proteinfamily, are directly involved in inflammation, vascular and autoimmunediseases, diabetes, Alzheimer, cancer and angiogenesis⁸²⁻⁸⁸. Proteinanalysis of CNV tissues revealed a dramatic reduction of the 35 kDaisoform of RAGE (the secreted isoform of RAGE describedpreviously^(89, 90)) n retina and choroid tissues from CNV lesiontreated by Lodamin. No detectable difference in the full size RAGE inthe retina was detected. In HMVEC cells, a dose dependant reduction ofthe full size receptor RAGE and its ligand HMGB-1 were obtained withTNP-470 (0.01-100 nM). HMGB-1 (Amphoterin) is a low molecular weightprotein with intracellular and extracellular regulatory activities thatis known to have pro-inflammatory and pro-angiogenicproperties^(87, 91). It has been demonstrated, in different models, thatRAGE ligands can induce a sustained activation of NFκB via MAPKactivation (e.g. ERK/p-ERK), enhance MCP-1 and TNFα production,upregulate VEGF in RPE, and increase MMPs activation^(84, 92-99). Allthese factors were shown to be suppressed by Lodamin in this CNV model,suggesting the possibility of additional indirect effects following thesuppression RAGE activation.

In previous reports it has been shown that S100A4, which is a RAGEligand and a member of S100 family, can covalently bind MetAp2¹⁰⁰, amolecular target of TNP-470. S100 is a family of nonubiquitouscytoplasmic Ca2+-binding proteins that are expressed in a wide varietyof cell types and are linked to many human pathologies¹⁰¹⁻¹⁰³. Thereduction of RAGE by TNP-470 provides a link between the knowninteractions of S 100-RAGE^(104, 105), S100-MetAp2¹⁰³ andTNP-470-MetAp2¹⁰⁶. It is likely that MetAp2 is associated with RAGEsignal and TNP-470 suppresses this signal in a direct or indirect way.

The current treatment for ARMD is primarily based on VEGF targeting.Because Lodamin can target broader molecular pathways, it maypotentially improve therapeutic outcomes. It is well established thatmany factors are playing a central role in the progression of thediseases in addition to VEGF. In addition, the specific structure ofLodamin, as a polymeric nanomicelle, enhances its accumulation in CNVlesions compared to the injection of free small molecule (as seen inFIG. 21) suggesting an ideal biodistribution via systemicadministration. This effect may be due to the Enhanced Permeability andRetention effect (EPR)¹⁰⁷ caused by the leaky vasculature in neovascularsites. Lodamin was well tolerated and showed minimal toxicity in theretina as indicated by histological analysis and ERG. Lastly, the slowrelease properties of Lodamin may minimize the dose and frequency oftreatments, which is particularly important for intravitreal therapies.The current study therefore supports the fact that Lodamin is anexcellent candidate for therapeutic treatment of ocular neovasculardiseases in humans.

The references cited herein and throughout the application areincorporated herein by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for the purposes of clarity ofunderstanding, one skilled in the art will easily ascertain that certainchanges and modifications may be practiced without departing from thespirit and scope of the appended claims.

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The invention claimed is:
 1. A composition formulated for oraladministration comprising a fumagillol derivative havinganti-proliferative activity, wherein said fumagillol derivative iscovalently linked to a block copolymer comprising a hydrophilic polymermoiety and a hydrophobic polymer moiety, wherein said block copolymer isa diblock copolymer comprising a PEG-PLA diblock copolymer havinghydrophilic PEG and hydrophobic PLA moieties, and wherein saidfumagillol derivative is covalently linked with the hydrophobic moietyof said block copolymer.
 2. The composition of claim 1, wherein saidfumagillol derivative comprises a derivative selected from the groupconsisting of 6-O—(N-chloroacetylcarbamoyl) fumagillol (TNP-470),6-O-(4-methoxyaniline)acetyl fumagillol;6-O-(3,4,5-trimethexyaniline)acetyl fumagillol;6-O-(4-(N,N-dimethylethoxy) aniline)acetyl fumagillol;6-O-(cyclopropylamino) acetyl fumagillol; 6-O-(cyclobutylamino)acetylfumagillol; 4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy-1,5 20dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol;4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol.
 3. Thecomposition of claim 1, wherein said composition comprises micelles. 4.A method of treating a condition involving or relying upon MetAP-2activity for its pathology, the method comprising administering an oralformulation comprising a fumagillol derivative having anti-proliferativeactivity, wherein said fumagillol derivative is covalently linked to ablock copolymer comprising a hydrophilic polymer moiety and ahydrophobic polymer moiety, wherein said block copolymer is a diblockcopolymer comprising a PEG-PLA diblock copolymer having hydrophilic PEGand hydrophobic PLA moieties, and wherein said fumagillol derivative iscovalently linked with the hydrophobic moiety of said block copolymer.5. The method of claim 4, wherein said fumagillol derivative comprises aderivative selected from the group consisting of6-O—(N-chloroacetylcarbamoyl) fumagillol (TNP-470),6-O-(4-methoxyaniline)acetyl fumagillol;6-O-(3,4,5-trimethexyaniline)acetyl fumagillol;6-O-(4-(N,N-dimethylethoxy) aniline)acetyl fumagillol;6-O-(cyclopropylamino) acetyl fumagillol; 6-O-(cyclobutylamino)acetylfumagillol; 4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy-1,5 20dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol;4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol.
 6. Themethod of claim 4, wherein the condition is selected from the groupconsisting of cancer, metastatic tumors, age-related maculardegeneration (AMD), and, ocular neovascularizations.
 7. The method ofclaim 4, wherein said composition comprises micelles.
 8. The method ofclaim 4, wherein said anti-proliferative composition has anti-tumoractivity.
 9. A method of making a diblock copolymer compositionformulated for oral administration and comprising a that hasanti-proliferative activity, the method comprising covalentlyconjugating said fumagillol derivative to a diblock copolymer comprisinga hydrophilic polymer moiety and a hydrophobic polymer moiety, whereinsaid block copolymer is a diblock copolymer comprising a PEG-PLA diblockcopolymer having hydrophilic PEG and hydrophobic PLA moieties, andwherein said fumagillol derivative is covalently linked with thehydrophobic moiety of said block copolymer.
 10. The method of claim 9,wherein said fumagillol derivative has anti-angiogenic activity.
 11. Themethod of claim 9, wherein said fumagillol derivative comprises aderivative selected from the group consisting of6-O—(N-chloroacetylcarbamoyl) fumagillol (TNP-470),6-O-(4-methoxyaniline)acetyl fumagillol;6-O-(3,4,5-trimethexyaniline)acetyl fumagillol;6-O-(4-(N,N-dimethylethoxy) aniline)acetyl fumagillol;6-O-(cyclopropylamino)acetyl fumagillol; 6-O-(cyclobutylamino)acetylfumagillol; 4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy-1,5 20dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol;4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol.
 12. Adiblock copolymer composition formulated for oral administration andcomprising a fumagillol derivative, said composition produced by themethod of claim
 9. 13. The diblock copolymer composition of claim 12,wherein said a fumagillol derivative has anti-proliferative activity.14. The diblock copolymer composition of claim 12, wherein saidfumagillol derivative comprises a derivative selected from the groupconsisting of 6-O—(N-chloroacetylcarbamoyl)fumagillol (TNP-470),6-O-(4-methoxyaniline)acetyl fumagillol;6-O-(3,4,5-trimethexyaniline)acetyl fumagillol;6-O-(4-(N,N-dimethylethoxy) aniline)acetyl fumagillol;6-O-(cyclopropylamino)acetyl fumagillol; 6-O-(cyclobutylamino)acetylfumagillol; 4-((cyclopropylamino)acetyl)oxy-2-(1,2-epoxy-1,5 20dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1 cyclohexanol;4-((cyclobutylamino)acetyl)oxy-2-(1,2-epoxy-1,5dimethyl-4-hexenyl)-3-methoxy-1-chloromethyl-1-cyclohexanol.